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Background: In conjunction with post placement in root-filled teeth with periapical healthy conditions, root canal retreatment may be performed to improve the seal of the root canal. Whether root canal retreatment for technical reasons (retreatments in teeth without apical periodontitis (AP)) results in lower frequency of AP is unknown., Objective: To examine whether there is a difference in frequency of AP between roots with root canals retreated for technical reasons, and roots with root canals not retreated before post placement, with a minimum follow-up of 5 years. Also, to examine changes in root filling quality following root canal retreatment for technical reasons., Methods: This retrospective study included radiographs of 441 root-filled roots without periapical radiolucencies at baseline, scheduled for post and core treatment. Follow-up data for a minimum of 5 years were available for 305 roots (loss to follow-up 30.8%), 46 of which were retreated for technical reasons. Two calibrated observers assessed root filling sealing quality and length, respectively, and periapical status according to the Periapical Index. The main outcome of the study, AP, was used as the dependent variable and all analyses were performed at root level., Results: The overall frequency of AP at follow-up was 13.8%. The difference in frequency of AP between retreated (4.3%) and nonretreated (15.4%) root canals was not statistically significant, p = .061. Analyses including only roots with preoperatively inadequate root filling quality showed a statistically significant difference (p = .017) between the two treatment groups (2.4% vs. 22.9%)., Conclusions: Root canal retreatment for technical reasons before post and core placement significantly reduces the frequency of AP in roots with inadequate root filling quality., (© 2024 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Marfan syndrome (MFS) is an autosomal dominant condition characterized by aortic aneurysm, skeletal abnormalities, and lens dislocation, and is caused by variants in the FBN1 gene. To explore causes of MFS and the prevalence of the disease in Iceland we collected information from all living individuals with a clinical diagnosis of MFS in Iceland (n = 32) and performed whole-genome sequencing of those who did not have a confirmed genetic diagnosis (27/32). Moreover, to assess a potential underdiagnosis of MFS in Iceland we attempted a genotype-based approach to identify individuals with MFS. We interrogated deCODE genetics' database of 35,712 whole-genome sequenced individuals to search for rare sequence variants in FBN1. Overall, we identified 15 pathogenic or likely pathogenic variants in FBN1 in 44 individuals, only 22 of whom were previously diagnosed with MFS. The most common of these variants, NM_000138.4:c.8038 C > T p.(Arg2680Cys), is present in a multi-generational pedigree, and was found to stem from a single forefather born around 1840. The p.(Arg2680Cys) variant associates with a form of MFS that seems to have an enrichment of abdominal aortic aneurysm, suggesting that this may be a particularly common feature of p.(Arg2680Cys)-associated MFS. Based on these combined genetic and clinical data, we show that MFS prevalence in Iceland could be as high as 1/6,600 in Iceland, compared to 1/10,000 based on clinical diagnosis alone, which indicates underdiagnosis of this actionable genetic disorder., (© 2023. The Author(s).)

We report a case of a man with a 30-year history of treatment-resistant hypertension, hydropoiesis, tachycardic spells and dysgeusia. Despite repeated visits to the emergency department and work-up in an out-patient clinic, the diagnosis was unknown. Three years prior to remittance to an endocrinologist, the hypertension worsened, and he developed diabetes type-II. Further work-up revealed a 3 cm extra-adrenal pheochromocytoma, a paraganglioma. After surgical removal of the tumor, he is without medication and symptom free. Pheochromocytoma and paraganglioma are rare causes of hypertension, estimated to explain 0.1-0.6% of all cases, but nonetheless an important diagnosis to make, due to serious side effects.

Predicting the pathogenicity of biallelic missense variants can be challenging. Here, we use a deficit of observed homozygous carriers of missense variants, versus an expected number in a set of 153,054 chip-genotyped Icelanders, to identify potentially pathogenic genotypes. We follow three missense variants with a complete deficit of homozygosity and find that their pathogenic effect in homozygous state ranges from severe childhood disease to early embryonic lethality. One of these variants is in CPSF3, a gene not previously linked to disease. From a set of clinically sequenced Icelanders, and by sequencing archival samples targeted through the Icelandic genealogy, we find four homozygous carriers. Additionally, we find two homozygous carriers of Mexican descent of another missense variant in CPSF3. All six homozygous carriers of missense variants in CPSF3 show severe intellectual disability, seizures, microcephaly, and abnormal muscle tone. Here, we show how the absence of certain homozygous genotypes from a large population set can elucidate causes of previously unexplained recessive diseases and early miscarriage., (© 2022. The Author(s).)

Introduction: A genetic counselling unit at Landspitali hospital (LSH) was established in 2006. Meanwhile, genetic testing has become an integral part of general health care. In this article we detail the outcome of genetic testing at the Department of Genetic and Molecular Medicine (DGM) at Landspitali over a five year period (2012-2017). Factors that were analyzed for the time period were: Number of patients, reason for referral, reason for genetic testing without genetic counselling and yield (proportion of positive tests) of genetic testing., Methods: Data was analysed from two medical record databases, Shire and Saga, used by the DGM in the time period., Results: The number of individuals coming for genetic counselling increased every year over the time period. Reasons for referral were cancer-related in two-thirds of cases. Other reasons for referral included various other familial disorders. Most common were autosomal dominant disorders like myotonic dystrophy, hypertrophic cardiomyopathy and autosomal recessive disorders like spinal muscular atrophy (SMA) and GM1-gangliosidosis. Most common reasons for genetic testing outside of the LSH GC unit was because of managable diseases like hemochromatosis and F5/Prothrombin-related thrombophilia. Yield of genetic testing was assessed for a) known mutation testing / carrier testing, b) single gene testing, c) gene panel testing and d) whole genome and whole exome sequencing. Known mutation testing was positive in 33% of cases and single gene testing in 46% of cases. The yield of gene panel testing for cancer was found to be lower (20%) than gene panel testing for other disorders (40%). The yield of whole exome and whole genome sequencing was 46%.

The study objective was to delineate the genetics of inherited retinal degenerations (IRDs) in Iceland, a small nation of 364.000 and a genetic isolate. Benefits include delineating novel pathogenic genetic variants and defining genetically homogenous patients as potential investigative molecular therapy candidates. The study sample comprised patients with IRD in Iceland ascertained through national centralized genetic and ophthalmological services at Landspitali, a national social support institute, and the Icelandic patient association. Information on patients' disease, syndrome, and genetic testing was collected in a clinical registry. Variants were reevaluated according to ACMG/AMP guidelines. Overall, 140 IRD patients were identified (point prevalence of 1/2.600), of which 70 patients had a genetic evaluation where two-thirds had an identified genetic cause. Thirteen disease genes were found in patients with retinitis pigmentosa, with the RLBP1 gene most common (n = 4). The c.1073 + 5G > A variant in the PRPF31 gene was homozygous in two RP patients. All tested patients with X-linked retinoschisis (XLRS) had the same possibly unique RS1 pathogenic variant, c.441G > A (p.Trp147X). Pathologic variants and genes for IRDs in Iceland did not resemble those described in ancestral North-Western European nations. Four variants were reclassified as likely pathogenic. One novel pathogenic variant defined a genetically homogenous XLRS patient group., (© 2021 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

There is an increased pressure to return results from research studies. In Iceland, deCODE Genetics has emphasised the importance of returning results to research participants, particularly the founder pathogenic BRCA2 variant; NM_000059.3:c.771_775del. To do so, they opened the website www.arfgerd.is . Individuals who received positive results via the website were offered genetic counselling (GC) at Landspitali in Reykjavik. At the end of May 2019, over 46.000 (19% of adults of Icelandic origin) had registered at the website and 352 (0.77%) received text message informing them about their positive results. Of those, 195 (55%) contacted the GC unit. Additionally, 129 relatives asked for GC and confirmatory testing, a total of 324 individuals. Various information such as gender and age, prior knowledge of the variant and perceived emotional impact, was collected. Of the BRCA2 positive individuals from the website, 74 (38%) had prior knowledge of the pathogenic variant (PV) in the family. The majority initially stated worries, anxiety or other negative emotion but later in the process many communicated gratitude for the knowledge gained. Males represented 41% of counsellees as opposed to less than 30% in the regular hereditary breast and ovarian (HBOC) clinic. It appears that counselling in clinical settings was more reassuring for worried counsellees. In this article, we describe one-year experience of the GC service to those who received positive results via the website. This experience offers a unique opportunity to study the public response of a successful method of the return of genetic results from research.

A comprehensive pedigree, usually provided by the counselee and verified by medical records, is essential for risk assessment in cancer genetic counseling. Collecting the relevant information is time-consuming and sometimes impossible. We studied the use of electronically ascertained pedigrees (EGP). The study group comprised women (n = 1352) receiving HBOC genetic counseling between December 2006 and December 2016 at Landspitali in Iceland. EGP's were ascertained using information from the population-based Genealogy Database and Icelandic Cancer Registry. The likelihood of being positive for the Icelandic founder BRCA2 pathogenic variant NM_000059.3:c.767_771delCAAAT was calculated using the risk assessment program Boadicea. We used this unique data to estimate the optimal size of pedigrees, e.g., those that best balance the accuracy of risk assessment using Boadicea and cost of ascertainment. Sub-groups of randomly selected 104 positive and 105 negative women for the founder BRCA2 PV were formed and Receiver Operating Characteristics curves compared for efficiency of PV prediction with a Boadicea score. The optimal pedigree size included 3° relatives or up to five generations with an average no. of 53.8 individuals (range 9-220) (AUC 0.801). Adding 4° relatives did not improve the outcome. Pedigrees including 3° relatives are difficult and sometimes impossible to generate with conventional methods. Pedigrees ascertained with data from pre-existing genealogy databases and cancer registries can save effort and contain more information than traditional pedigrees. Genetic services should consider generating EGP's which requires access to an accurate genealogy database and cancer registry. Local data protection laws and regulations have to be addressed.

DNA damage assays have various limitations in types of lesions detected, sensitivity, specificity and samples that can be analyzed. The Northern Lights Assay (NLA) is based on 2D Strandness-Dependent Electrophoresis (2D-SDE), a technique that separates nucleic acids based on length, strandness, structure and conformation changes induced by damage. NLA is run on a microgel platform in 20-25 min. Each specimen is analyzed in pairs of non-digested DNA to detect single- and double-stranded breaks (DSBs) and Mbo I-digested DNA to detect other lesions. We used NLA to evaluate DNA in solution and isolated from human cells treated with various genotoxic agents. NLA detected and distinguished between single- and DSBs, interstrand and intrastrand DNA crosslinks, and denatured single-stranded DNA. NLA was sufficiently sensitive to detect biologically relevant amount of DNA damage. NLA is a versatile, sensitive and simple method for comprehensive and simultaneous analysis of multiple types of damage, both in purified DNA and in DNA isolated from cells and body fluids. NLA can be used to evaluate DNA quality in biosamples, monitor complex molecular procedures, assess genotoxicity, diagnose genome instability, facilitate cancer theranostics and in basic nucleic acids research.

We describe a system for horizontal 1D or 2D PAGE comprising an apparatus and microgels. There is no buffer outside the gel, making handling and sample loading easy. Specially designed electrodes on all four sides allow 2D electrophoresis without gel rotation. Electrophoresis is completed within 20 min and sensitivity is in the subnanogram range. The system is temperature controlled for speed, denaturation of nucleic acid molecules and maintaining molecules single-stranded. The system allows characterization of structure, conformation and damage in complex nucleic acid preparations. Besides quick 1D PAGE, 2D applications include characterization of efficiency of complex molecular procedures, checking quality of biosamples and detecting DNA damage in cells and body fluids. The system should also run protein gels.

While pedigree drawing software is often utilised in genetic services, the use of genealogical databases in genetic counselling is unusual. This is mainly because of the unavailability of such databases in most countries. Electronically generated pedigrees used for cancer genetic counselling in Iceland create pedigrees that automatically incorporate information from a large, comprehensive genealogy database and nation-wide cancer registry. The aim of this descriptive qualitative study was to explore counsellees' experiences of genetic services, including family history taking, using these electronically generated pedigrees. Four online focus groups with 19 participants were formed, using an asynchronous posting method. Participants were encouraged to discuss their responses to questions posted on the website by the researcher. The main themes arising were motivation, information and trust, impact of testing and emotional responses. Most of the participants expressed trust in the method of using electronically generated pedigrees, although some voiced worries about information safety. Many experienced worry and anxiety while waiting for results of genetic testing, but limited survival guilt was noted. Family communication was either unchanged or improved following genetic counselling. The use of electronically generated pedigrees was well received by participants, and they trusted the information obtained via the databases. Age did not seem to influence responses. These results may be indicative of the particular culture in Iceland, where genealogical information is well known and freely shared. Further studies are needed to determine whether use of similar approaches to genealogical information gathering may be acceptable elsewhere.

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

Background: Ethical considerations are increasingly important in medicine. We aimed to determine the mode and extent of teaching of ethics in training programs in clinical chemistry and laboratory medicine., Methods: We developed an on-line survey of teaching in areas of ethics relevant to laboratory medicine. Reponses were invited from directors of training programs who were recruited via email to leaders of national organizations., Results: The survey was completed by 80 directors from 24 countries who directed 113 programs. The largest numbers of respondents directed postdoctoral training of scientists (42%) or physicians (33%), post-masters degree programs (33%), and PhD programs (29%). Most programs (82%) were 2years or longer in duration. Formal training was offered in research ethics by 39%, medical ethics by 31%, professional ethics by 24% and business ethics by 9%. The number of reported hours of formal training varied widely, e.g., from 0 to >15h/year for research ethics and from 0 to >15h for medical ethics. Ethics training was required and/or tested in 75% of programs that offered training. A majority (54%) of respondents reported plans to add or enhance training in ethics; many indicated a desire for online resources related to ethics, especially resources with self-assessment tools., Conclusion: Formal teaching of ethics is absent from many training programs in clinical chemistry and laboratory medicine, with heterogeneity in the extent and methods of ethics training among the programs that provide the training. A perceived need exists for online training tools, especially tools with self-assessment components., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Membrane transporters enable efficient cellular metabolism, aid in nutrient sensing, and have been associated with various diseases, such as obesity and cancer. Genome-scale metabolic network reconstructions capture genomic, physiological, and biochemical knowledge of a target organism, along with a detailed representation of the cellular metabolite transport mechanisms. Since the first reconstruction of human metabolism, Recon 1, published in 2007, progress has been made in the field of metabolite transport. Recently, we published an updated reconstruction, Recon 2, which significantly improved the metabolic coverage and functionality. Human metabolic reconstructions have been used to investigate the role of metabolism in disease and to predict biomarkers and drug targets. Given the importance of cellular transport systems in understanding human metabolism in health and disease, we analyzed the coverage of transport systems for various metabolite classes in Recon 2. We will review the current knowledge on transporters (i.e., their preferred substrates, transport mechanisms, metabolic relevance, and disease association for each metabolite class). We will assess missing coverage and propose modifications and additions through a transport module that is functional when combined with Recon 2. This information will be valuable for further refinements. These data will also provide starting points for further experiments by highlighting areas of incomplete knowledge. This review represents the first comprehensive overview of the transporters involved in central metabolism and their transport mechanisms, thus serving as a compendium of metabolite transporters specific for human metabolic reconstructions.

This brief report aims to give an overview of the history and current status of clinical genetics services in Iceland and specific genetic counseling considerations for Iceland's population. Presently, there are two part time medical geneticists and one full time genetic counselor with an MSc education from Cardiff, within the Department of Genetic and Molecular Medicine, based in Iceland's only tertiary healthcare facility, Landspitali, the National University Hospital. An oncologist (20 %) also contributes to the cancer genetic counseling service. In addition, a pediatric medical geneticist has a 25 % appointment at the Children's Hospital. No other health care organization offers genetic counseling, and there are no private genetic counseling services.

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ∼2× more reactions and ∼1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.

Background: The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids., Methods: We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis., Results: The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples., Conclusions: Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis., (© 2013 American Association for Clinical Chemistry)

Unlabelled: The use of electronic genealogical databases facilitates the construction of accurate and extensive pedigrees for potential use in genetic services. Genealogy databases can be linked to specific disease databases, such as cancer registries, in order to increase the accuracy of pedigrees used, and inform the genetic risk assessment. To review the published literature on the use of genealogy databases to construct pedigrees for risk assessment in genetic health service, a systematic literature search was undertaken using 12 combined search terms to identify all relevant published articles., Data Sources: EbscoHost, PubMed, Web of Science, Ovid and the "grey literature", as well as the reference lists of identified studies. Of 1,035 titles identified, two papers described a study on the use of genealogy databases in cancer risk assessment and two were discussion papers. While authors of the four papers described the potential use of genealogy databases in clinical genetic services, such use has not been adequately investigated and further research is required.

Inborn errors of metabolism (IEMs) are hereditary metabolic defects, which are encountered in almost all major metabolic pathways occurring in man. Many IEMs are screened for in neonates through metabolomic analysis of dried blood spot samples. To enable the mapping of these metabolomic data onto the published human metabolic reconstruction, we added missing reactions and pathways involved in acylcarnitine (AC) and fatty acid oxidation (FAO) metabolism. Using literary data, we reconstructed an AC/FAO module consisting of 352 reactions and 139 metabolites. When this module was combined with the human metabolic reconstruction, the synthesis of 39 acylcarnitines and 22 amino acids, which are routinely measured, was captured and 235 distinct IEMs could be mapped. We collected phenotypic and clinical features for each IEM enabling comprehensive classification. We found that carbohydrate, amino acid, and lipid metabolism were most affected by the IEMs, while the brain was the most commonly affected organ. Furthermore, we analyzed the IEMs in the context of metabolic network topology to gain insight into common features between metabolically connected IEMs. While many known examples were identified, we discovered some surprising IEM pairs that shared reactions as well as clinical features but not necessarily causal genes. Moreover, we could also re-confirm that acetyl-CoA acts as a central metabolite. This network based analysis leads to further insight of hot spots in human metabolism with respect to IEMs. The presented comprehensive knowledge base of IEMs will provide a valuable tool in studying metabolic changes involved in inherited metabolic diseases.

Purpose: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries., Methods: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors., Results: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events., Conclusion: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%.

A girl presented with congenital arthrogryposis, intellectual disability and mild bone-related dysmorphism. Molecular workup including the NimbleGen Human CGH 2.1M platform revealed a 1.13 Mb de novo microdeletion on chromosome 12q13.13 of paternal origin. The deletion contains 33 genes, including AAAS, AMRH2, and RARG genes as well as the HOXC gene cluster. At least one gene, CSAD, is expressed in fetal brain. The deletion partially overlaps number of reported benign CNVs and pathogenic duplications. This case appears to represent a previously unknown microdeletion syndrome and possibly the first description in humans of a disease phenotype associated with copy loss of HOXC genes., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: A defense system against transposon activity in the human germline based on PIWI proteins and piRNA has recently been discovered. It represses the activity of LINE-1 elements via DNA methylation by a largely unknown mechanism. Based on the dispersed distribution of clusters of piRNA genes in a strand-specific manner on all human chromosomes, we hypothesized that this system might work preferentially on local and proximal sequences. We tested this hypothesis with a methylation-associated SNP (mSNP) marker which is based on the density of C-T transitions in CpG dinucleotides as a surrogate marker for germline methylation., Results: We found significantly higher density of mSNPs flanking piRNA clusters in the human genome for flank sizes of 1-16 Mb. A dose-response relationship between number of piRNA genes and mSNP density was found for up to 16 Mb of flanking sequences. The chromosomal density of hypermethylated LINE-1 elements had a significant positive correlation with the chromosomal density of piRNA genes (r = 0.41, P = 0.05). Genome windows of 1-16 Mb containing piRNA clusters had significantly more hypermethylated LINE-1 elements than windows not containing piRNA clusters. Finally, the minimum distance to the next piRNA cluster was significantly shorter for hypermethylated LINE-1 compared to normally methylated elements (14.4 Mb vs 16.1 Mb)., Conclusions: Our observations support our hypothesis that the piRNA-PIWI system preferentially methylates sequences in close proximity to the piRNA clusters and perhaps physically adjacent sequences on other chromosomes. Furthermore they suggest that this proximity effect extends up to 16 Mb. This could be due to an unknown localization signal, transcription of piRNA genes near the nuclear membrane or the presence of an unknown RNA molecule that spreads across the chromosome and targets the methylation directed by the piRNA-PIWI complex. Our data suggest a region specific molecular mechanism which can be sought experimentally.

A potential relationship between transposon-derived repeats (TDR) and human germline methylation is of biological importance since many genes are flanked by TDR and methylation could affect the expression of nearby genes. Furthermore, DNA methylation has been suggested as a global defense mechanism against genome instability threatened by TDR. We studied the correlation between the density of HapMap methyl-associated SNPs (mSNPs), a marker of germline methylation, and proportion of TDR. After correcting for confounding variables, we found a negative correlation between proportion of Alu repeats and mSNP density for 125-1000 kb windows. Similar results were found for the most active subgroup of repeats. In contrast, a negative correlation between proportion of L1 repeats and mSNP density was found only in the larger 1000 kb windows. Using methylation data on germ cells (sperm) from the Human Epigenome Project, we found a lower proportion of Alu repeats adjacent (3-15 kb) to hypermethylated amplicons. On the contrary, there was a higher proportion of L1 repeats in the 3-5 kb of sequence flanking hypermethylated amplicons but not in the 10-15 kb flanks. Our data indicate a differential response to the major repeat families and that DNA methylation is unlikely to be a uniform global defense system against all TDR. It appears to play a role for the L1 subgroup, with sequences adjacent to L1 repeats methylated in response to their proximity. In contrast, sequences adjacent to Alu repeats appear to be hypomethylated, arguing against a role of methylation in germline defense against those elements., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objectives: To assess pregnant women's knowledge and understanding of first trimester prenatal screening (nuchal translucency, maternal serum free beta-human chorionic gonadotrophin and pregnancy-associated plasma-protein-A), to evaluate the impact of a new information booklet and investigate the effects of education and experiential knowledge of congenital disabilities on the perceived likelihood of accepting prenatal screening., Design: A quasi-experimental quantitative study with a self-completion questionnaire., Setting: Five different maternity care clinics in Iceland., Population: Expectant mothers in first trimester of pregnancy (n = 379)., Material and Methods: Expectant mothers were divided into two groups, an intervention and a control group, both receiving traditional care and information. The intervention group additionally received an information booklet about prenatal screening and diagnosis., Main Outcome Measures: Women's knowledge score of prenatal screening. The correlation between education, knowledge score, experiential knowledge of congenital disabilities, and the likelihood of accepting prenatal screening., Results: More than half of the women (57%) believed they received sufficient information to make an informed decision about screening. Knowledge scores were significantly higher for the intervention group (with mean 4.8 compared with 3.7 on a 0-8 scale, p < 0.0001). Those with higher scores were more likely to accept screening (p < 0.0001). Women with experiential knowledge of congenital anomalies in their own families were more likely to accept prenatal screening (p = 0.017)., Conclusions: Various factors, e.g. experiential knowledge, education and information about prenatal screening affect the likelihood of participation in prenatal screening programs. More information results in better knowledge and higher uptake rate.

We describe a case of acute intermittent porphyria in a woman who presented repeatedly with abdominal pain. Porphyrias are caused by decreased enzyme activity in the heme biosynthetic pathway leading to overproduction of heme precursors if demand increases. This can cause symptoms such as abdominal pain, nausea and vomiting, constipation, tachycardia and hypertension. Treatment includes removal of causative factors, administration of carbohydrates or hemin to reduce the production of heme precursors as well as symptomatic treatment.

Inter-individual and regional variability in recombination rates cannot be fully explained by the DNA sequence itself. Epigenetic mechanisms might be one additional factor affecting recombination. A biochemical approach to studying human germline methylation is difficult. We used the density of the 434,198 nonredundant methylation-associated SNPs (mSNPs) in the derived allele HapMap data set as a surrogate marker for germline DNA methylation. We validated our methodology by demonstrating that the mSNP density confirmed known patterns of genomic methylation, including the hypermutability of methylated cytosine and hypomethylation of CpG islands. Using this approach, we found a genome-wide positive correlation between germline methylation and regional recombination rate (500-kb windows: r = 0.622, P < 10(-15)). This remained significant with multiple correlations correcting for sequence features known to affect recombination, such as GC content and CpG dinucleotides (500-kb windows: r = 0.172, P < 10(-15)). Using the ENCODE data set for increased resolution, we found a positive correlation between germline DNA methylation and recombination rate (50-kb windows: r = 0.301, P = 0.002). This correlation was further strengthened when corrected for sequence features affecting recombination (50-kb windows: r = 0.445, P < 0.0001). In the Human Epigenome Project data set there was increased DNA methylation in regions within recombination hot spots in male germ cells (0.632 vs. 0.557, P = 0.007). The relationship we observed between germline DNA methylation and recombination could be explained in two ways that are not mutually exclusive: DNA methylation could indicate preferred sites for recombination, or methylation following recombination could inhibit further recombination, perhaps by being part of the enigmatic molecular pathway mediating crossover interference.

System analysis of metabolic network reconstructions can be used to calculate functional states or phenotypes. This provides tools to study the metabolic effects of genetic and epigenetic properties, such as dosage sensitivity. We used the genome-scale reconstruction of human metabolism (Recon 1) to analyze the effect of nine known or predicted imprinted genes on metabolic phenotypes. Simulations of maternal deletion of ATP10A indicated an anabolic metabolism consistent with the known clinical phenotypes of obesity. The abnormal expression of the other genes affected fewer subsections of metabolism consistent with a lack of established clinical phenotypes. We found that four of nine genes had metabolic effect as predicted by the Haig's parental conflict theory.

Laboratory Medicine organizations and their professional members have a goal and responsibility to benefit the health and wellbeing of the patients and communities they serve. Newer genetics and biochemical techniques raise significant issues of community concern, impacting on privacy, informed consent, access to and retention of samples and information. Balance may be required to ensure protection of individual rights against potential benefits to the broader community. While many national organizations may already have appropriate policies addressing various ethics issues, there is a need for an international framework to assist those nations that have not yet developed such policies, as well as to enable alignment of existing national policies. We have proposed a generic ethics framework, incorporating a hierarchy of four fundamental guiding principles: autonomy, justice, non-maleficence and beneficence. Proposals or issues requiring policy development can be considered and tested against this hierarchy, resulting in the development of policy and positions consistent with the above framework, acceptable to all participating stakeholders.

Lipid peroxidation of polyunsaturated fatty acids (PUFA) generates reactive products that may cause DNA damage. To examine the possible relationship between DNA damage in peripheral blood mononuclear cells (PBMC) and the concentration of PUFA in red blood cells (RBC), endogenous DNA strand breaks, formamidopyrimidine DNA glycosylase (FPG) sites, and hydrogen peroxide (H2O2) sensitive sites were evaluated by the comet assay in blood samples from 98 Icelandic women. Fatty acid composition of RBC was analyzed by gas chromatography. Endogenous DNA strand breaks in PBMC correlated positively with the concentration of total PUFA, total n-3 PUFA, docosahexaenoic acid, linoleic acid, oleic acid, and palmitic acid in RBC. However, there was no association between FPG sites or H(2)O(2) sensitive sites in DNA in PBMC and the concentration of total PUFA or total saturated fatty acid in RBC. As there was no association between oxidative DNA damage or sensitivity of DNA to oxidative stress and the concentration of PUFA in RBC, the positive association between endogenous DNA strand breaks in PBMC and the concentration of total PUFA in RBC is probably not related to oxidative stress.

We investigated the role of the 5'-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Delta3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Delta5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Delta5MSD mutant and a double mutant (DM) (Delta5MSD and Delta3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Delta5MSD was dispensable in FOS cells, and the DM had the same REE as the Delta3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors.

Dendritic cells (DC) are the main producers of the cytokine IL-12p70, through which they play a direct role in the development of IFN-gamma-secreting Th1 cells, costimulation of CTL differentiation and NK-cell activation. In contrast, IL-10, which is also produced by DC, negatively regulates IL-12 production. IL-12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL-12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL-12p70 secretion by monocyte-derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL-12p70, presence of the IL10 high genotype restricted IL-12p70 production in these individuals. These observations provide a further genetic control of IL-12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

Transposon-derived repeats (TDR) represent approximately 50% of the human genome. A transposon suppression system has been proposed to explain why transposon-derived repeats (TDR) seldom cause mutations in humans. If this system is based on DNA methylation, a correlation might exist between amount of TDR adjacent to genes and frequency of coding sequence mutations due to m5C deaminations. To test this hypothesis we selected 385 genes based on availability of accurate information on their genome structure and mutation patterns (at least 10 mutations described in the Human Gene Mutation Database (HGMD)). The CENSOR program was used to estimate amount and class of TDR for the gene region and an arbitrarily selected 1 KB from each end. We assumed all C --> T transitions to be possible 5-methylcytosine-associated mutations (MAM) and calculated the number and proportion of MAM in the 385 genes. If there is a strong correlation between methylation of certain CpX dinuclecotides and TDR we might be able to detect it despite limitations of available data for this analysis. We found statistically significant correlations between: i) TDR and number of MAM in genes (r = 0.118, p = 0.02), ii) SINE-TDR and proportion of CpG --> TpG (r = 0.11, p = 0.03); limited to MIR elements only (r = 0.14, p = 0.006), and iii) LINE-TDR and proportion of CpT --> TpT (r = 0.166, p = 0.04). The group of genes with no TDR had a statistically significant lower proportion of MAM (184/479, 0.38 vs. 6466/14524, 0.46; p = 0.009) with differences noted for CpA --> TpA (35/479, 0.073 vs. 1380/11474; p = 0.003). In addition, CpT --> TpT were least common in genes with no TDR (8/479, 0.017), intermediate in genes with TDR in genomic sequence but not mRNA (337/11474, 0.029) and most common in genes with TDR within mature mRNA (121/3050, 0.040; p for trend = 0.003). Our data suggest that TDR adjacent to genes may sometimes influence methylation of cytosines in coding sequences to a degree that it affects mutation patterns. These observations should be followed up with further database analysis and biochemical studies.

We describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.

PUFA are susceptible to oxidation. However, the chain-reaction of lipid peroxidation can be interrupted by antioxidants. Whether an increased concentration of PUFA in the body leads to decreased antioxidant capacity and/or increased consumption of antioxidants is not known. To elucidate the relationship between plasma total antioxidant capacity (TAC), the concentration of antioxidant vitamins, and the proportion of PUFA in red blood cells (RBC), plasma TAC was measured by a Trolox equivalent antioxidant capacity assay in blood samples from 99 Icelandic women. Concentrations of tocopherols and carotenoids in the plasma were determined by HPLC, and the FA composition of RBC total lipids was analyzed by GC. Plasma TAC and the plasma concentration of alpha-tocopherol correlated positively with the proportion of total n-3 PUFA, 20:5n-3, and 22:6n-3 in RBC, whereas the plasma lycopene concentration correlated negatively with the proportion of total n-3 PUFA and 20:5n-3. On the other hand, plasma TAC correlated negatively with the proportion of n-6 PUFA in RBC. Plasma TAC also correlated positively with the plasma concentration of alpha-tocopherol, alcohol consumption, and age. Both the plasma concentration of alpha-tocopherol and age correlated positively with the proportion of n-3 PUFA in RBC; however, n-3 PUFA contributed independently to the correlation with plasma TAC. Because the proportion of n-3 PUFA in RBC reflects the consumption of n-3 PUFA, these results suggest that dietary n-3 PUFA do not have adverse effects on plasma TAC or the plasma concentration of most antioxidant vitamins.

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.

Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells.

Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials. The genus Populus is the internationally accepted model for molecular tree biology. We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons. We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence. A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs. The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation. The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation. These data will also valuable for functional genomic efforts in Arabidopsis.

DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples.

We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 x 10(7) copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.

Expression of iduronate-2-sulfatase (IDS) from three different promoters in four retroviral vectors was studied in peripheral blood lymphocytes from patients with Hunter syndrome (PBL(MPS)), i.e., the LTR in vectors L2SN and L2, avian beta-actin promoter in LB2, and the CMV early promoter in LNC2. PBL(MPS) were exposed to packaging cell supernatant resulting in transduction frequencies ranging 10-fold from 5 to 49%. Surprisingly, IDS activities were equally high in all transduced lymphocyte populations: 515 U/mg/h in PBL(MPS)-L2SN, 734 in PBL(MPS)-LB2, 352 in PBL(MPS)-L2, and 389 in PBL(MPS)-LNC2 compared to controls (<10 in PBL(MPS)-LXSN or PBL(MPS)). The half-life of endocytosed IDS in PBL(MPS) was 1.9 days. However, the level of lymphocyte IDS activity from proviral expression was found to be only a fraction of the total, a large portion being derived from reuptake of enzyme from murine packaging cells, i.e., a "second source" of enzyme. Therefore, measurement of transgene lysosomal enzyme soon after exposure of target cells to vector supernatant may yield a gross overestimate of long-term transgene expression by transduced cells. Nevertheless, patient fibroblasts cocultured with transduced PBL(MPS) had reduced (35)SO(4)-GAG accumulation, levels similar to those of normal fibroblasts. These studies revealed a broadly applicable phenomenon: cells can be charged with a lysosomal enzyme to levels much higher than those found in nature. By "supercharging" cells with a lysosomal protein (or other molecule bearing the mannose-6-phosphate ligand), such cells may be exploited as vehicles for systemic delivery of therapeutic or diagnostic agents., (Copyright 2000 Academic Press.)

We describe a family with four members, a mother, two sons, and a daughter, who show clinical features consistent with X linked Alport syndrome. The two males presented with additional features including mental retardation, dysmorphic facies with marked midface hypoplasia, and elliptocytosis. The elliptocytosis was not associated with any detectable abnormalities in red cell membrane proteins; red cell membrane stability and rigidity was normal on ektacytometry. Molecular characterisation suggests a submicroscopic X chromosome deletion encompassing the entire COL4A5 gene. We propose that the additional abnormalities found in the affected males of this family are attributable to deletion or disruption of X linked recessive genes adjacent to the COL4A5 gene and that this constellation of findings may represent a new X linked contiguous gene deletion syndrome.

We observed a family in which two boys were diagnosed with Alport syndrome, elliptocytosis, and mental retardation and carried a large deletion of the Xq22.3-q23 region, encompassing the COL4A5 gene. This suggests the possibility of a new contiguous gene syndrome. In an attempt to characterize the genes contributing to this complex phenotype, we have isolated a gene encoding a new long-chain acyl-CoA synthetase (FACL4 or LACS4) from the region deleted in these patients. Among several ESTs identified by searching the human gene map database maintained at the National Center for Biotechnology Information, using the map position as a query, only one was deleted in the patients. RACE products containing the entire ORF were subsequently generated. Northern blot analysis showed a 5-kb mRNA expressed in several tissues except for liver and lung. Brain shows a longer transcript, possibly reflecting the use of a brain-specific upstream ATG start codon. FACL4 encodes a predicted protein product of 670 amino acids (711 in brain), with a remarkable level of conservation compared to the rat acyl-CoA synthetases ACS4 and brain-specific ACS3 protein sequences. We are investigating the possibility that the absence of this enzyme may play a role in the development of mental retardation or other signs associated with Alport syndrome in the family., (Copyright 1998 Academic Press.)

The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q.

To explore the feasibility of ex vivo lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II), we evaluated retrovirus-mediated gene transfer of the iduronate-2-sulfatase (IDS) coding sequence into peripheral blood lymphocytes from enzyme-deficient individuals (PBLMPS). Moloney murine leukemia virus-derived retroviral vectors were constructed by inserting the IDS cDNA under transcriptional regulation of the long terminal repeat (LTR) (in vector L2SN) or the cytomegalovirus (CMV) early promoter (vector LNC2). High-titer virus-producer cells were generated using amphotropic PA317 packaging cells. After 3 days of in vitro stimulation of T lymphocytes with anti-CD3 antibody and interleukin-2 (IL-2), PBLMPS were transduced once on each of the next 3 days. Seven to 21 days later, cultured PBLMPS were evaluated for gene transfer and IDS specific activity. Heterogeneous populations of L2SN-transduced PBLMPS had high levels of IDS enzyme activity (456 U/mg per hr +/- SD 292) despite a gene transfer efficiency of 5% or less. Owing to overexpression of IDS in that percentage of PBLMPS successfully transduced, IDS activity was increased above the deficiency found in patients with Hunter syndrome (< 20 U/mg per hr) to a level comparable with that of normal individuals (mean activity of uncultured normal leukocytes 807 U/mg per hr; SD 252). Reduced 35SO4-glucosaminoglycan (GAG) accumulation was observed in PBLMPS that had been transduced with L2SN, or when PBLMPS were grown in medium that had been "conditioned" by growth of L2SN-transduced cells. This latter result indicated that metabolic cross-correction occurred by means of intercellular enzyme transfer. These studies of retrovirus-mediated expression and metabolic correction, finding near-normal levels of IDS in cultured PBLMPS and metabolic correction, demonstrate the potential for treatment of mild, nonneuropathic Hunter syndrome by means of ex vivo lymphocyte gene therapy.

We constructed and tested several retroviral vectors containing abbreviated purine nucleoside phosphorylase (PNP) genes in the reverse orientation, a strategy compatible with transduction of intron-containing genes. We observed two types of deletions in these vectors after one round of replication: (i) Deletions flanked by direct repeats with one copy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) Deletions due to fortuitous splice sites in the PNP complementary strand. Two splice donor sites and three splice acceptor sites were identified in a 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysis of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The putative PNP polyadenylation signal and a truncated 117-bp 3' flank were recovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank. Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor signals in the complementary strand were inactivated. A retroviral vector LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reverse orientation, was transduced intact in 23% (5/22) of clones after one round of replication and in 87% (20/23) of clones after a second round of replication from two primary virus producer clones. Directed mutagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.

Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C-->T, severe phenotype); P358R (1073C-->G, severe); R468W (1402C-->T, mild); P469H (1406C-->A, mild); and Y523C (1568A-->G, mild). Non-sense mutations were identified in two patients: R172X (514C-->T, severe) and Q389X (1165C-->T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (> 95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395 C-->G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented.