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STUDY QUESTIONCan risks associated with novelties in assisted reproduction technologies (ARTs) be assessed in a systematic and structured way?SUMMARY ANSWERAn ART-specific risk assessment tool has been developed to assess the risks associated with the development of novelties in ART (EuroGTP II-ART).WHAT IS KNOWN ALREADYHow to implement new technologies in ART is well-described in the literature. The successive steps should include testing in animal models, executing pre-clinical studies using supernumerary gametes or embryos, prospective clinical trials and finally, short- and long-term follow-up studies on the health of the offspring. A framework categorizing treatments from experimental through innovative to established according to the extent of the studies conducted has been devised. However, a systematic and standardized methodology to facilitate risk evaluation before innovations are performed in a clinical setting is lacking.STUDY DESIGN, SIZE, DURATIONThe EuroGTP II-ART risk assessment tool was developed on the basis of a generic risk assessment algorithm developed for tissue and cell therapies and products (TCTPs) in the context of the project ‘Good Practices for demonstrating safety and quality through recipient follow-up European Good Tissue and cells Practices II (EuroGTP II)’. For this purpose, a series of four meetings was held in which eight ART experts participated. In addition, several tests and simulations were undertaken to fine-tune the final tool.PARTICIPANTS/MATERIALS, SETTING, METHODSThe three steps comprising the EuroGTP II methodology were evaluated against its usefulness and applicability in ART. Ways to improve and adapt the methodology into ART risk assessment were agreed and implemented.MAIN RESULTS AND THE ROLE OF CHANCEAssessment of the novelty (Step 1), consisting of seven questions, is the same as for other TCTPs. Practical examples were included for better understanding. Identification of potential risks and consequences (Step 2), consisting of a series of risks and risk consequences to consider during risk assessment, was adapted from the generic methodology, adding more potential risks for processes involving gonadic tissues. The algorithm to score risks was also adapted, giving a specific range of highest possible risk scores. A list of strategies for risk reduction and definition of extended studies required to ensure effectiveness and safety (Step 3) was also produced by the ART experts, based on generic EuroGTP II methodology. Several explanations and examples were provided for each of the steps for better understanding within this field.LIMITATIONS, REASONS FOR CAUTIONA multidisciplinary team is needed to perform risk assessment, to interpret results and to determine risk mitigation strategies and/or next steps required to ensure the safety in the clinical use of novelties.WIDER IMPLICATIONS OF THE FINDINGSThis is a dynamic tool whose value goes beyond assessment of risk before implementing a novel ART in clinical practice, to re-evaluate risks based on information collected during the process.STUDY FUNDING / COMPETING INTEREST(S)This study was called EUROGTP II and was funded by the European Commission (Grant agreement number 709567). The authors declare no competing interests concerning the results of this study.

IntroductionDue to an increase in multicentric paediatric clinical trials after 2007 following the EU Paediatric Regulation, there has been a substantial increase in the number of feasibility questionnaires (FQ). Paediatric clinical trial conduct has made unique advances through the conect4children (c4c) pan-European network, funded by the Innovative Medicines Initiative (IMI2). We examined the role of a national, centralized coordination center in Belgium to pre-fill feasibility questionnaires and quality control responses from sites.MethodsThis report describes the 4-year learnings of prefilling and performing quality control for FQ by the Belgian Network representative. Additional information has been included from a broad survey sent to sites in the Belgian Network, of which 13 of the 15 sites have responded.ResultsPIs are confronted by between 10 to 50 FQ requests per year, each taking at average 60 minutes. The number of redundant questions asked by sponsors is on average 43% of the FQ. Of the 112 completed feasibilities, approximately 82% required quality control adjustments by our national coordinating center. Inconsistencies were primarily found in PIs’ report of experience, number of paediatric studies conducted, recruitment estimates and site qualifications. With a centralized and repetitive collection of FQ, a prefill of 65% of the requested information and potential corrections can be performed. A time reduction of 10 to 46% is estimated when a FQ is facilitated through the national representative.ConclusionsThe increase in paediatric clinical trials has substantially burdened sites within Belgium. Quality control and adjustments by a national central organization could be beneficial to increase feasibility quality and efficiency.

IntroductionDue to the Paediatric Regulation in 2007, the number of paediatric clinical trials within Europe has substantially increased. Consequently, potential sites for paediatric clinical trials were overrun by trial opportunities, infrastructure pressure and their limited experience. To support sites and facilitate research, the pan-European network conect4children (c4c) was established and funded by the Innovative Medicines Initiative (IMI2). In order to attain sustainable research networks, site requirements innovation needs require further investigation.MethodsA questionnaire was conducted within the c4c Belgian Clinical trial network. An inquiry was made into the role of a national hub and other topics for support. Due to multiple responses per site, answers were grouped per site.ResultsOf the 15 connected sites, we received 32 responses coming from 13 unique sites. Within the Belgian network, around 4 (30%) of sites do not have a paediatric clinical trial unit to support trials. All sites agreed or strongly agreed that a national hub is useful for the site to conduct clinical trials. The majority namely 11 (84%) of sites identified human resources as a core improvement need for sites, specifically finding dedicated clinical research nurses and finding time for principal investigators (PI) to perform study tasks aside from clinical work. A strong need for financing of infrastructure from 10 (76%) of sites is acknowledged for consultation areas, imaging, and a biobank structure.ConclusionsIn order to foster sustainable development of new medicines in paediatric diseases, site need’s must to be taken into account and prioritized. The primary need is in human resources and financing of infrastructure.

Aim: The European Network of Excellence for Paediatric Clinical Research, known as the TEDDY Network, carried out a survey to determine the capacity and competence of paediatric centres to perform research studies. Methods: A cross-sectional, web-based pilot survey was conducted from October 2016 to April 2017 with paediatric clinical research centres in 11 countries: Albania, Austria, Belgium, Denmark, Iceland, Ireland, Italy, Norway, Spain, Switzerland and the United Kingdom. All were registered with the TEDDY Network database. Results: We approached 107 centres and 63 provided data on their experiences and expertise in paediatric clinical trials. Four groups of performance indicators were identified, referring to scientific experience, trial readiness, trial competence, regulatory issues, ethics and patients. Most centres were actively involved in paediatric clinical research: 53 centres (84.1%) had received funds for more than five paediatric studies in the last 5 years, and 42 (66.7%) had a specific clinical trial unit and dedicated study coordinators. We concluded that the European centres we studied had the capability and capacity to conduct paediatric trials, but there was still room for improvement, including enhanced collaboration. Conclusion: This pilot survey demonstrated that there is potential for performing paediatric trials across Europe, but improvements are possible.

Contains fulltext : 242783.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To capture the early effects of the coronavirus disease 2019 (COVID-19) pandemic on pediatric clinical research. STUDY DESIGN: Pediatric clinical research networks from 20 countries and 50 of their affiliated research sites completed two surveys over one month from early May to early June 2020. Networks liaised with their affiliated sites and contributed to the interpretation of results through pan-European group discussions. Based on first detection dates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), countries formed 1 early detecting and 1 late detecting cluster. We tested the hypothesis that this clustering influenced clinical research. RESULTS: Research sites were first impacted by the pandemic in mid-March 2020 (March 16 ± 10 days, the same date as lockdown initiation; P = .99). From first impact up until early June, site initiation and feasibility analysis processes were affected for >50% of the sites. Staff were redirected to COVID-19 research for 44% of the sites, and 75.5% of sites were involved in pediatric COVID-19 research (only 6.3% reported COVID-19 cases in their other pediatric trials). Mitigation strategies were used differently between the early and late detecting country clusters and between countries with and without a pediatric COVID-19 research taskforce. Positive effects include the development of teleworking capacities. CONCLUSIONS: Through this collaborative effort from pediatric research networks, we found that pediatric trials were affected and conducted with a range of unequally applied mitigations across countries during the pandemic. The global impact might be greater than captured. In a context where clinical research is increasingly multinational, this report reveals the importance of collaboration between national networks.

Mannan-Binding lectin (MBL) is a serum lectin and an important constituent of the innate immune system. Processes linked to the innate immune response have previously been implicated in Alzheimer's disease (AD). MBL is associated with blood vessels in the brain and AD patients demonstrate lower MBL levels in the cerebrospinal fluid compared to controls. We investigated six single nucleotide polymorphisms, linked to MBL deficiency, in the corresponding MBL2 gene in AD patients and controls. Two MBL2 haplotypes, LXP and LYQ, were significantly associated with AD risk (OR = 1.6, p = 0.01 and OR = 1.5, p = 0.02, respectively). The present study is the first investigating MBL2 genotypes and haplotypes in relation to AD. Our findings support that the MBL2 gene impact the disease risk.

Background A common and serious complication of cardiac surgery prompting early detection and intervention is cardiac surgery-associated acute kidney injury (CSA-AKI). Urinary chitinase 3-like protein 1 (UCHI3L1) was found to predict AKI associated with critical illness in adults. Our aims were therefore to evaluate whether UCHI3L1 can also be used to predict AKI associated with elective cardiac surgery in adults, and to compare this predictive ability with that of urinary neutrophil gelatinase-associated lipocalin (UNGAL), more frequently assessed early serum creatinine (SCr) measurements, and various two-biomarker panels. Methods This was a single-centre prospective cohort study at the eight-bed cardiac surgery ICU of Ghent University Hospital. AKI was diagnosed and classified according to the Kidney Disease|Improving Global Outcomes definitions for the diagnosis and staging of AKI, which are based on SCr and urine output (UO). Of the 211 enrolled elective cardiac surgery patients, we included 203 patients who had no AKI pre-operatively and at time of post-operative ICU admission (t1) in the primary endpoint analysis (i.e. AKI stage ≥1 within 48 h after t1), while 210 patients without AKI stage ≥2 pre-operatively and at t1 were included in the secondary endpoint analysis (i.e. AKI stage ≥2 within 12 h after t1). Systemic and/or urine concentrations of Cr, CHI3L1 and NGAL were measured more frequently than SCr in routine early post-operative ICU practice. UO was monitored hourly in the ICU. Results Within 48 h after t1, 46.8% of the patients had developed AKI (70.5% stage 1, 20.0% stage 2 and 9.5% stage 3). In the early post-operative period, only SCr was a good predictor of AKI within 48 h after t1 (primary endpoint). SCHI3L1 combined with either UCHI3L1 or UNGAL was a good predictor of AKI stage ≥2 within 12 h after t1 (secondary endpoint). However, SCr and its absolute difference from pre-operative to early measures after surgery outperformed these combinations. Conclusions We found that more frequent assessment of the functional biomarker SCr in the early post-operative ICU period (first 4 h) after elective cardiac surgery in adult patients had good to excellent predictive value for CSA-AKI, indicating that routine SCr assessment must become more frequent in order to detect AKI more early. This performance was in contrast with the inadequate predictive value of the urinary renal stress or damage biomarkers UCHI3L1 and UNGAL. Electronic supplementary material The online version of this article (doi:10.1186/s13613-017-0251-z) contains supplementary material, which is available to authorized users.

Host genetic factors have previously been found to act as determinants of differential susceptibility to major infectious diseases. It is less clear whether such polymorphisms may also impose on pathogen recognition in mucosal overgrowth conditions such as bacterial vaginosis, an anaerobic overgrowth condition characterised by the presence of a vaginal biofilm consisting of the Gram-positive anaerobes Gardnerella vaginalis and Atopobium vaginae. We selected 34 single nucleotide polymorphisms pertaining to 9 genes involved with Toll-like receptor-mediated pathogen recognition and/or regulation (LBP, CD14, TLR1, TLR2, TLR4, TLR6, MD2, CARD15 and SIGIRR) and assessed in a nested case-control study their putative association with bacterial vaginosis, as diagnosed by Gram staining, and with the vaginal carriage of A. vaginae and G. vaginalis, as determined by species-specific PCR, among 144 pregnant women. Carriage of G. vaginalis during early pregnancy was associated with the -1155A>G substitution in the promoter region of the MD2 gene (p=0.041). The presence of A. vaginae during the first half of the pregnancy was significantly associated with the CD14 intron 2 1342G>T (p=0.039), the TLR1 exon 4 743A>G (p=0.038), and the CARD15 exon 4 14772A>T (p=0.012) polymorphisms, and marginally significantly associated with the LBP exon13 26842C>T (p=0.056), the CD14 promoter -260C>T (p=0.052), and the TLR1 promoter -7202A>G (p=0.062) polymorphisms. However, no association between gene polymorphisms and bacterial vaginosis as such could be documented. Our data suggest that some degree of genetic susceptibility involving pathogen recognition may occur with the key bacterial vaginosis organism, A. vaginae.

Background Acute kidney injury (AKI) occurs frequently and adversely affects patient and kidney outcomes, especially when its severity increases from stage 1 to stages 2 or 3. Early interventions may counteract such deterioration, but this requires early detection. Our aim was to evaluate whether the novel renal damage biomarker urinary chitinase 3-like protein 1 (UCHI3L1) can detect AKI stage ≥2 more early than serum creatinine and urine output, using the respective Kidney Disease | Improving Global Outcomes (KDIGO) criteria for definition and classification of AKI, and compare this to urinary neutrophil gelatinase-associated lipocalin (UNGAL). Methods This was a translational single-center, prospective cohort study at the 22-bed surgical and 14-bed medical intensive care units (ICU) of Ghent University Hospital. We enrolled 181 severely ill adult patients who did not yet have AKI stage ≥2 based on the KDIGO criteria at time of enrollment. The concentration of creatinine (serum, urine) and CHI3L1 (serum, urine) was measured at least daily, and urine output hourly, in the period from enrollment till ICU discharge with a maximum of 7 ICU-days. The concentration of UNGAL was measured at enrollment. The primary endpoint was the development of AKI stage ≥2 within 12 h after enrollment. Results After enrollment, 21 (12 %) patients developed AKI stage ≥2 within the next 7 days, with 6 (3 %) of them reaching this condition within the first 12 h. The enrollment concentration of UCHI3L1 predicted the occurrence of AKI stage ≥2 within the next 12 h with a good AUC-ROC of 0.792 (95 % CI: 0.726–0.849). This performance was similar to that of UNGAL (AUC-ROC of 0.748 (95 % CI: 0.678–0.810)). Also, the samples collected in the 24-h time frame preceding diagnosis of the 1st episode of AKI stage ≥2 had a 2.0 times higher (95 % CI: 1.3–3.1) estimated marginal mean of UCHI3L1 than controls. We further found that increasing UCHI3L1 concentrations were associated with increasing AKI severity. Conclusions In this pilot study we found that UCHI3L1 was a good biomarker for prediction of AKI stage ≥2 in adult ICU patients.

Summary l-ficolin (also called ficolin-2, P35 or hucolin) is a soluble pattern recognition molecule of suspected importance in anti-microbial immunity. It activates the lectin pathway of complement and acts as an opsonin. l-ficolin, encoded by the FCN2 gene, recognizes microbial polysaccharides and glycoconjugates rich in GlcNAc or GalNAc. We report here data concerning four single nucleotide polymorphisms (SNPs) of the FCN2 gene and their relationship to l-ficolin serum concentrations. There are two pairs of SNPs in linkage disequilibrium: ss32469536 (located in promoter) with rs7851696 (in exon 8) and ss32469537 (promoter) with ss32469544 (exon 8). We selected groups possessing low or high serum l-ficolin concentrations (≤ 2·8 µg/ml or ≥ 4·5 µg/ml, respectively) from Polish children suffering from recurrent respiratory infections (n = 146). Low l-ficolin levels were associated with variant alleles for ss32469536 and rs7851696 and normal alleles for ss32469537 and ss32469544. Conversely, high l-ficolin levels were associated with variant alleles of ss32469537 and ss32469544. FCN2 genotyping should be a valuable additional tool for disease association studies.

We report on a girl with moderate developmental delay and mild dysmorphic features. Cytogenetic investigations revealed a de novo interstitial deletion at the proximal dark band on the long arm of chromosome 7 (7q21.1-q21.3) in all analyzed G-banded metaphases of lymphocytes and fibroblasts. Fluorescence in situ hybridization (FISH) and molecular studies defined the breakpoints at 7q21.11 and 7q21.3 on the paternal chromosome 7, with the proximal deletion breakpoint between the elastin gene (localized at 7q11.23) and D7S2517, and the distal breakpoint between D7S652 and the COL1A2 gene (localized at 7q21.3-q22.1). Deletions of interstitial segments at the proximal long arm of chromosome 7 at q21 are relatively rare. The karyotype-phenotype correlation of these patients is reviewed and discussed. The clinical findings of patients with a deletion at 7q21 significantly overlap with those of patients with maternal uniparental disomy of chromosome 7 (matUPD(7)) and Silver-Russell syndrome (SRS, OMIM 180860). Therefore, 7q21 might be considered a candidate chromosomal region for matUPD(7) and SRS.

As a key component of the complement system, mannose-binding lectin (MBL) is one of the linchpins of innate immunity. It is, therefore, not surprising that MBL2 genetic variants affecting the quantity and activity of the MBL protein in serum have been associated with increased susceptibility to infection and autoimmune diseases, and with poorer prognostic outcomes. This enhanced risk is particularly the case for children and immunosuppressed patients, especially when immunity is further compromised by coexistent primary or secondary immune deficiencies. In several disease areas, such as sepsis, cystic fibrosis, and recurrent childhood infections, the association between low MBL-producing allelic variants and disease risk and/or severity is particularly strong. It is here that the use of MBL testing and replacement therapy has reached the threshold of personalized medicine. The role of MBL in health and disease, advances in MBL testing methodologies and key areas for possible applications of MBL replacement therapy are reviewed.

In order to estimate the contribution of mutations at the fibrillin-1 locus (FBN1) to classical Marfan syndrome (MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11 mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven FBN1 mutation did not differ from the others with respect to the presence of major cardiac, ocular, and skeletal manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole, locus for MFS.

Classic Ehlers-Danlos syndrome (EDS) is characterized by fragile and hyperextensible skin, atrophic scarring, and joint hypermobility. Mutations in the COL5A1 and the COL5A2 gene encoding the alpha1(V) and the alpha2(V) chains, respectively, of type V collagen have been shown to cause the disorder, but it is unknown what proportion of classic EDS patients carries a mutation in these genes. We studied fibroblast cultures from 48 patients with classic EDS by SDS-PAGE for the presence of type V collagen defects. An abnormal collagen pattern was detected in only 2 out of 48 cell lines, making this a poor method for routine diagnostic evaluation. A total of 42 out of 48 (88%) patients were heterozygous for an expressed polymorphic variant in COL5A1. cDNA from 18 (43%) of them expressed only one COL5A1 allele. In 37 patients, the COL5A1/A2 genes were then analyzed by SSCP and conformation sensitive gel electrophoresis (CSGE). A total of 26 patients that were mutation-negative after SSCP/CSGE screening were reanalyzed by dHPLC. In addition, 11 other patients were analyzed by dHPLC only. In total, 17 mutations leading to a premature stop codon and five structural mutations were identified in the COL5A1 and the COL5A2 genes. In three patients with a positive COL5A1 null-allele test, no causal mutation was found. Overall, in 25 out of 48 patients (52%) with classic EDS, an abnormality in type V collagen was confirmed. Variability in severity of the phenotype was observed, but no significant genotype-phenotype correlations emerged. The relatively low mutation detection rate suggests that other genes are involved in classic EDS. We excluded the COL1A1, COL1A2, and DCN gene as major candidate genes for classic EDS, since no causal mutation in these genes was found in a number of patients who tested negative for COL5A1 and COL5A2.

Skin hyperelasticity, tissue fragility with atrophic scars, and joint hypermobility are characteristic for the classical type of Ehlers-Danlos syndrome (EDS). The disease is usually inherited as an autosomal dominant trait; however, recessive mode of inheritance has been documented in tenascin-X-deficient EDS patients. Mutations in the genes coding for collagen alpha1(V) chain (COL5A1), collagen alpha2(V) chain (COL5A2), tenascin-X (TNX), and collagen alpha1(I) chain (COL1A1) have been characterized in patients with classical EDS, thus confirming the suspected genetic heterogeneity. Recently, we described a patient with severe classical EDS due to a Gly1489Glu substitution in the alpha1(V) triple-helical domain who was, in addition, heterozygous for a disease-modifying Gly530Ser substitution in the alpha1(V) NH(2)-terminal domain [Giunta and Steinmann, 2000: Am. J. Med. Genet. 90:72-79; Steinmann and Giunta, 2000: Am. J. Med. Genet. 93:342]. Here, we report on a 4-year-old boy with mild classical EDS, born to healthy consanguineous Turkish parents; the mother presented a soft skin, while the father had a normal thick skin. Ultrastructural analysis of the dermis revealed in the patient the typical "cauliflower" collagen fibrils, while in both parents variable moderate aberrations were seen. Mutation revealed the presence of a homozygous Gly530Ser substitution in the alpha1(V) collagen chains in the patient, while both parents were heterozygous for the same substitution. An additional mutation in either the COL5A1 and COL5A2 genes was excluded. Furthermore, haplotype analysis with polymorphic microsatellite markers excluded linkage to the genes coding for alpha3(V) collagen (COL5A3), tenascin-X (TNX), thrombospondin-2 (THBS2), and decorin (DCN). These new findings support further our previous hypothesis that the heterozygous Gly530Ser substitution is disease modifying and now suggest that in the homozygous state it is disease causing.

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin disorder. In this report we have investigated an Italian child affected with recessive DEB (RDEB) and demonstrated that he was homozygous for the mutation R226X in the type VII collagen gene (COL7A1), leading to absence of type VII collagen at the dermal-epidermal junction. There was no family history of inherited skin blistering but the child's father was affected by Marfan syndrome, an autosomal dominant connective tissue disorder that results from mutations in the fibrillin-1 gene (FBN1). Analysis of this gene showed that the RDEB patient and his father were both heterozygous for a novel FBN1 mutation, C1971Y. This mutation affects one of the six obligate cysteine residues within one of the calcium-binding epidermal growth factor-like regions of the protein. At the age of 2-years the RDEB patient showed signs of early aortic dilatation, suggesting that he is likely to develop a Marfan syndrome phenotype in the future. This is a unique case of these two coexisting inherited disorders.

Classical Ehlers-Danlos syndrome (EDS) is characterized by skin hyperelasticity, joint hypermobility, increased tendency to bruise, and abnormal scarring. Mutations in type V collagen, a regulator of type I collagen fibrillogenesis, have been shown to underlie this type of EDS. However, to date, mutations have been found in only a limited number of patients, which suggests genetic heterogeneity. In this article, we report two unrelated patients with typical features of classical EDS, including excessive skin fragility, in whom we found an identical arginine--cysteine substitution in type I collagen, localized at position 134 of the alpha1(I) collagen chain. The arginine residue is highly conserved and localized in the X position of the Gly-X-Y triplet. As a consequence, intermolecular disulfide bridges are formed, resulting in type I collagen aggregates, which are retained in the cells. Whereas substitutions of glycine residues in type I collagen invariably result in osteogenesis imperfecta, substitutions of nonglycine residues in type I collagen have not yet been associated with a human disease. In contrast, arginine--cysteine substitutions in type II collagen have been identified in a variety of chondrodysplasias. Our findings show that mutations in other fibrillar collagens can be causally involved in classical EDS and point to genetic heterogeneity of this disorder.

SummaryAlthough >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.

A boy with features suggesting the diagnosis of Wiedemann-Rautenstrauch syndrome (WRS) or neonatal progeroid syndrome is presented. Abnormal findings included a generalized virtual absence of subcutaneous fat, sparse scalp hair, prominence of veins and muscles, a large and persistent anterior fontanelle and facial dysmorphism (triangular aged face, prominent eyes and scalp veins). Until now, only 13 cases (including one prenatal diagnosis) of this syndrome have been described. Since the borderlines of this syndrome are not very exact, we reviewed the previous reports in order to further delineate this rare syndrome.

We report two sibs with severe, progressively deforming osteogenesis imperfecta (OI) and homozygosity by descent for a glycine 751 to serine substitution in the alpha2(I) collagen chain due to a G to A transition in the COL1A2 gene. The parents, who were first cousins, and two elder sibs were heterozygous for the mutation and presented mild clinical manifestations of OI. Collagen studies on cultured fibroblasts from one of the probands and from the father showed that cells from the homozygote produced only mutant, unstable collagen I, whereas cells from the heterozygote produced both normal and mutant collagen I. This family represents an exceptional example of autosomal recessive OI, caused by homozygosity for a missense mutation in collagen I.

A single base mismatch was detected by single-strand conformation polymorphism (SSCP) of the collagen type III gene in a patient with Ehlers-Danlos syndrome type IV. The patient's fibroblasts secreted both normal and slowly migrating type III procollagen molecules. Two-dimensional CNBr peptide mapping suggested that the defect was localised in the CB9 peptide or the C-propeptide region of the alpha 1 (III)-chain. Analysis of a set of restriction-endonuclease-digested fragments of an amplified cDNA sequence encoding CB9, identified a single-strand conformation polymorphism and localized it within a region of 79 bp corresponding to the carboxyl-terminal end of the CB9 peptide of the alpha 1(III)-chain. DNA sequence analysis demonstrated that the patient was heterozygous for a point mutation converting G to T at base pair 3440 of the collagen alpha 1(III) cDNA resulting in the substitution of glycine with valine at amino acid position 1009 of the alpha 1(III)-chain. The mutation in this patient lies within a region of mutations at the carboxyl-terminal end of the type III collagen alpha-helix which all produce a severe "acrogeric" form of EDS IV.

Two patients with osteoporosis pseudoglioma syndrome are described. Both are single children, born to nonconsanguineous, healthy parents. The first patient, a 17-year-old girl, had serious visual impairment since birth. She is severely dwarfed and has major skeletal deformities resulting in inability to walk since age 2 years. The second patient is an 18-year-old girl with unilateral neonatal blindness, short stature and deformities, mainly of pelvis and lower limbs. She has been able to walk with support up to now. The clinical and radiological findings in these 2 patients reflect the clinical variability of the condition. Results of collagen studies in both patients are normal and differentiate this condition clearly from severe osteogenesis imperfecta, which it resembles. © 1993 Wiley-Liss, Inc.

The clinical and biochemical observations in a patient with a mild form of Ehlers-Danlos syndrome (EDS) type IV are described. The patient's skin fibroblasts produced markedly diminished amounts of type III collagen. SDS-polyacrylamide gel electrophoresis of collagens produced by cells obtained from other, non-cutaneous tissues showed two forms of collagen alpha 1(III) chains, a normal and a slow migrating, mutant form. Further analysis confirmed that the type III collagen molecules containing mutant alpha chains which were overmodified had a lower thermal stability and were poorly secreted into the extracellular medium. The protein defect was mapped by in situ cyanogen bromide digestion and was located in alpha 1(III) CB9, the C-terminal peptide of the collagen triple helix. This study shows that non-cutaneous connective tissues can be a useful source for the study of type III collagen defects in patients with EDS type IV.

A girl with a mild sporadic osteochondrodysplasia (OCD) similar to hypochondroplasia but with significant short stature is reported. She has been followed clinically between the ages of 9 months and 14 years. Growth remained normal throughout childhood with stature evolving about 3.5 SDs under the mean for age. By 8 years of age gradually appearing acanthosis nigricans (AN) in the neck and flanks was histopathologically confirmed. It provided the new incentive to search for specific FGFR3 mutations associated with this dermatologic abnormality. This resulted in the identification of the 1948A>C transversion predicting the K650Q missense substitution in the FGFR3 protein. Besides the expansion of the phenotypic spectrum of FGFR3-related OCDs to HCH with AN, this observation underscores the continuing adverse effect of this specific mutation upon the normal inhibitory signaling of the receptor at least in epidermal cells.

We report on a girl with moderate developmental delay and mild dysmorphic features. Cytogenetic investigations revealed a de novo interstitial deletion at the proximal dark band on the long arm of chromosome 7 (7q21.1-q21.3) in all analyzed G-banded metaphases of lymphocytes and fibroblasts. Fluorescence in situ hybridization (FISH) and molecular studies defined the breakpoints at 7q21.11 and 7q21.3 on the paternal chromosome 7, with the proximal deletion breakpoint between the elastin gene (localized at 7q11.23) and D7S2517, and the distal breakpoint between D7S652 and the COL1A2 gene (localized at 7q21.3-q22.1). Deletions of interstitial segments at the proximal long arm of chromosome 7 at q21 are relatively rare. The karyotype-phenotype correlation of these patients is reviewed and discussed. The clinical findings of patients with a deletion at 7q21 significantly overlap with those of patients with maternal uniparental disomy of chromosome 7 (matUPD(7)) and Silver-Russell syndrome (SRS, OMIM 180860). Therefore, 7q21 might be considered a candidate chromosomal region for matUPD(7) and SRS.

Ehlers-Danlos syndrome (EDS) type VIIC, or dermatosparactic type, is a recessively inherited connective tissue disorder characterized, among other symptoms, by an extreme skin fragility resulting from mutations inactivating ADAMTS-2, an enzyme excising the aminopropeptide of procollagens type I, II, and III. All previously described mutations create premature stop codons leading to a marked reduction in the level of mRNA. In this study, we analyzed the ADAMTS2 cDNA sequences from five patients displaying clinical and/or biochemical features consistent with a diagnosis of either typical or potentially mild form of EDS type VIIC. Three different alterations were detected in the two patients with typical EDS type VIIC. The first patient was homozygous for a genomic deletion causing an in-frame skipping of exons 3-5 in the transcript. In the second patient, the allele inherited from the mother lacks exon 3, generating a premature stop codon, whereas the paternal allele has a genomic deletion resulting in an in-frame skipping of exons 14-16 at the mRNA level. Although the exons 3-5 or 14-16 encode protein domains that have not been previously recognized as crucial for ADAMTS-2 activity, the aminoprocollagen processing was strongly impaired in vitro and in vivo, providing evidence for the requirement of these domains for proper enzyme function. The three other patients with a phenotype with some resemblance to EDS type VIIC only had silent and functionally neutral variations also frequently found in a normal population.

Item does not contain fulltext Marfan syndrome (MFS) is an autosomal dominant disorder with a prevalence of 2-3 per 10 000 individuals. Symptoms range from skeletal overgrowth, cutaneous striae to ectopia lentis and aortic dilatation leading to dissection. Prenatal diagnosis was until recently mainly performed in familial cases by linkage analysis. However, mutation detection has become available with thorough screening methods. The phenotypic variability observed in MFS makes reproductive options difficult, as molecular diagnosis cannot predict clinical severity of the disease. Data are presented on 15 prenatal and/or preimplantation genetic diagnoses (PGD) in nine families, originating from Belgium, the Netherlands, Spain and France. In four families data from linkage analysis were used, whereas in five other families the causative FBN1 mutation was characterised. Four PGD cycles in two couples led to one ongoing pregnancy. In addition, two amniocenteses and nine chorionic villus (CV) samplings were performed. In five pregnancies an affected fetus was diagnosed. In one of them, the couple chose to continue the pregnancy and an affected child was born, whereas the other four couples decided to terminate the pregnancy. It is expected that the greater availability of mutation testing of the FBN1 gene will increase requests for prenatal diagnosis. PGD appears to be an acceptable alternative for couples facing ethical reproductive dilemmas.

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin disorder. In this report we have investigated an Italian child affected with recessive DEB (RDEB) and demonstrated that he was homozygous for the mutation R226X in the type VII collagen gene (COL7A1), leading to absence of type VII collagen at the dermal-epidermal junction. There was no family history of inherited skin blistering but the child's father was affected by Marfan syndrome, an autosomal dominant connective tissue disorder that results from mutations in the fibrillin-1 gene (FBN1). Analysis of this gene showed that the RDEB patient and his father were both heterozygous for a novel FBN1 mutation, C1971Y. This mutation affects one of the six obligate cysteine residues within one of the calcium-binding epidermal growth factor-like regions of the protein. At the age of 2-years the RDEB patient showed signs of early aortic dilatation, suggesting that he is likely to develop a Marfan syndrome phenotype in the future. This is a unique case of these two coexisting inherited disorders.

Achondrogenesis II-hypochondrogenesis and severe spondyloepiphyseal dysplasia congenita (SEDC) are lethal forms of dwarfism caused by dominant mutations in the type II collagen gene (COL2A1). To identify the underlying defect in seven cases with this group of conditions, we used the combined strategy of cartilage protein analysis and COL2A1 mutation analysis. Overmodified type II collagen and the presence of type I collagen was found in the cartilage matrix of all seven cases. Five patients were heterozygous for a nucleotide change that predicted a glycine substitution in the triple helical domain (G313S, G517V, G571A, G910C, G943S). In all five cases, analysis of cartilage type II collagen suggested incorporation of the abnormal α1(II) chain in the extracellular collagen trimers. The G943S mutation has been reported previously in another unrelated patient with a strikingly similar phenotype, illustrating the possible specific effect of the mutation. The radiographically less severely affected patient was heterozygous for a 4 bp deletion in the splice donor site of intron 35, likely to result in aberrant splicing. One case was shown to be heterozygous for a single nucleotide change predicted to result in a T1191N substitution in the carboxy-propeptide of the proα1(II) collagen chain. Study of the clinical, radiographic, and morphological features of the seven cases supports evidence for a phenotypic continuum between achondrogenesis II-hypochondrogenesis and lethal SEDC and suggests a relationship between the amount of type I collagen in the cartilage and the severity of the phenotype. Keywords: type II collagen disorders; achondrogenesis II-hypochondrogenesis; spondyloepiphyseal dysplasia congenita; COL2A1

Editor—Hypochondroplasia is a mild, autosomal dominant skeletal dysplasia. The relative dearth of specific clinical manifestations and the absence of pathognomonic radiographic features often make the diagnosis of hypochondroplasia difficult.1-4 Short limbed dwarfism is rarely recognised before the age of 2 years and is usually mild with heights up to the low normal range. Muscular body build, macrocephaly with mild frontal bossing, and lumbar hyperlordosis are frequently reported. The radiographic features are variable and can be almost normal in mildly affected subjects.4 They most typically include no change or decrease in the interpedicular distance from the first to the fifth lumbar vertebral bodies, anteroposterior shortening of the lumbar pedicles, short iliac bones with flat acetabular roof, small sacrosciatic notches, short tubular bones, short and broad femoral necks, and relative elongation of the distal or proximal portion of the fibula.2 4 Proof that achondroplasia and hypochondroplasia are allelic disorders came with the discovery that both conditions map to the distal short arm of chromosome 4.5 6 Subsequently, mutation analysis of the FGFR3 gene, located in the 4p16.3 region, showed a recurrent mutation (N540K) in several unrelated hypochondroplasia patients.7 8 Recently, two novel mutations in the same region of the FGFR3 gene causing hypochondroplasia have been identified: N540T in a Dutch family and I538V in a Swedish kindred.9 10 In some sporadic patients and …

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

Clinical and biochemical findings in 5 unrelated patients with osteogenesis imperfecta (OI) with a serine for glycine substitution in the alpha2(I) collagen chain are presented. The data are compared to other serine substitutions in collagen type I. Findings show that the phenotypic severity of serine for glycine substitutions in the alpha2(I) collagen chain is region dependent similar to the observations for the alpha1(I) collagen chain, and that so-called 'lethal' and 'non-lethal' domains in the alpha1 and alpha2 collagen chains do not necessarily correspond.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are autosomal dominant osteochondrodysplasias that result in mild to severe short-limb dwarfism and early-onset osteoarthrosis. PSACH and some forms of MED result from mutations in the gene for cartilage oligomeric matrix protein (COMP; OMIM 600310 [http://www3.ncbi.nlm. nih.gov:80/htbin-post/Omim/dispmim?600310]). We report the identification of COMP mutations in an additional 14 families with PSACH or MED phenotypes. Mutations predicted to result in single-amino acid deletions or substitutions, all in the region of the COMP gene encoding the calmodulin-like repeat elements, were identified in patients with moderate to severe PSACH. We also identified within this domain a missense mutation that produced MED Fairbank. In two families, one with mild PSACH and the second with a form of MED, we identified different substitutions for a residue in the carboxyl-terminal globular region of COMP. Both the clinical presentations of these two families and the identification of COMP-gene mutations provide evidence of phenotypic overlap between PSACH and MED. These data also reveal a role for the carboxyl-terminal domain in the structure and/or function of COMP.

Pretibial epidermolysis bullosa is a rare form of dominant dystrophic epidermolysis bullosa. The disease was diagnosed after considerable delay in a large Belgian family and was remarkable for its late age at onset and its misleading clinical presentation in the proband, which strongly resembled keratosis lichenoides chronica. Both recessively and dominantly inherited forms of dystrophic epidermolysis bullosa have been shown to be linked to the collagen type VII gene, COL7A1. Two-point linkage analysis with two intragenic polymorphisms (PvuII, AluI) in COL7A1 was performed. Strong genetic linkage between the disease in this family and COL7A1 was demonstrated by a lod score of 4.45 (theta = 0) for the AluI polymorphism. The observed intrafamilial variability of clinical phenotypes contradicts the presently proposed classification of dominantly inherited dystrophic epidermolysis bullosa.