Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were

COL1A1 C-propeptide cleavage site mutation causes high bone mass, bone fragility and jaw lesions: a new cause of gnathodiaphyseal dysplasia?

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant condition characterized by bone fragility, irregular bone mineral density (BMD) and fibro-osseous lesions in the skull and jaw. Mutations in Anoctamin-5 (ANO5) have been identified in some cases. We aimed to identify the causative mutation in a family with features of GDD but no mutation in ANO5, using whole exome capture and massive parallel sequencing (WES). WES of two affected individuals (a mother and son) and the mother's unaffected parents identified a mutation in the C-propeptide cleavage site of COL1A1. Similar mutations have been reported in individuals with osteogenesis imperfecta (OI) and paradoxically increased BMD. C-propeptide cleavage site mutations in COL1A1 may not only cause 'high bone mass OI', but also the clinical features of GDD, specifically irregular sclerotic BMD and fibro-osseous lesions in the skull and jaw. GDD patients negative for ANO5 mutations should be assessed for mutations in type I collagen C-propeptide cleavage sites.

Next-generation sequencing: a frameshift in skeletal dysplasia gene discovery.

In the last decade, huge breakthroughs in genetics-driven by new technology and different statistical approaches-have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis.

A report of three patients with MMP2 associated hereditary osteolysis.

Osteolysis syndromes are rare hereditary disorders characterized by destruction and resorption of affected bones. The current study adds three new patients from two unrelated consanguineous families with a severe form of inherited osteolysis. Clinical examination, radiological, biochemical, ultrastructural and molecular studies were conducted. Clinical and radiological studies suggested the diagnosis of Torg-Winchester syndrome. The three affected patients were homozygous for novel MMP2 gene mutations which confirmed the diagnosis. Our patients are the first to be reported from Egypt thus, supporting the pan ethnic nature of the disease.

HMGB1 as a predictor of infarct transmurality and functional recovery in patients with myocardial infarction.

OBJECTIVES: High-mobility group box 1 (HMGB1) protein is an innate danger signal for the initiation of host defence and tissue repair. The aim of this study was to analyse serum HMGB1 concentration and its correlation with infarct transmurality and functional recovery in patients with ST-elevation (STEMI) and non-ST-elevation myocardial infarction (NSTEMI). DESIGN: We prospectively examined patients with first-time STEMI (n = 46) or NSTEMI (n = 49), treated according to current guidelines. Contrast-enhanced cardiac magnetic resonance imaging was performed 2-4 days after infarction for the estimation of infarct transmurality and was repeated after 6 months for the estimation of residual left ventricular function. HMGB1 was measured 2-4 days after infarction. RESULTS: High-mobility group box 1 concentration was related to infarct size and to residual ejection fraction in patients with STEMI (r(2) = 0.81 and r(2) =0.40, respectively, P < 0.001 for both) and NSTEMI (r(2) = 0.74 and r(2) = 0.25, respectively, P < 0.001 for both). Receiver operating characteristic (ROC) curve-derived cut-off values of 6.2 and 5.9 ng mL(-1) for patients with STEMI and NSTEMI, respectively, were predictive of infarct transmurality greater than 75% (STEMI: area under the curve (AUC) = 0.93, standard error (SE) = 0.04, 95% confidence interval (CI) = 0.81-0.98; NSTEMI: AUC = 0.96, SE = 0.04, 95% CI = 0.86-0.99). HMGB1 cut-off values of 7.2 and 6.4 ng mL(-1) for patients with STEMI and NSTEMI, respectively, were predictive of residual ejection fraction 6 months after myocardial infarction (MI) (STEMI: AUC = 0.81, SE = 0.07, 95% CI = 0.66-0.91; NSTEMI: AUC = 0.81, SE = 0.09, 95% CI = 0.68-0.91). CONCLUSION: High-mobility group box 1 serum levels represent a highly valuable surrogate marker for infarct transmurality and for the prediction of residual left ventricular function after MI.

Radial artery thrombosis following transradial coronary angiography: incidence and rationale for treatment of symptomatic patients with low-molecular-weight heparins.

BACKGROUND: Transradial access for diagnostic and therapeutic coronary angiography gains more and more popularity because of its advantages over the femoral approach, enhancing patient comfort, reducing bleeding complications and duration of hospital stay. However, these benefits are overshadowed by an increased rate of radial artery (RA) occlusion. There are little data regarding the exact incidence, potential predictors and outcome of post-procedural RA occlusions. Furthermore, there is no clear evidence for the optimal treatment of this complication. METHODS: In a single-centre prospective observational study, 488 consecutive patients were evaluated by ultrasound the day after transradial cardiac catheterization for signs of RA occlusion. Symptomatic patients with sonographically identified radial artery thrombosis underwent treatment with low-molecular-weight heparin (LMWH) for 4 weeks. Asymptomatic patients did not receive anticoagulation therapy. The primary endpoint was the patency rate of the radial artery at 4 weeks of follow-up. RESULTS: Radial artery thrombosis was found in 51 of 488 (10.5%) patients 1 day after transradial cardiac catheterization. 30 (58.8%) patients showed symptoms on access site, whereas 21 (41.2%) did not show any symptoms. After 4 weeks, 26 (86.7%) of the symptomatic patients showed a partial or complete recanalization of the radial artery after treatment with LMWH, compared with 4 (19.1%) of the asymptomatic patients without anticoagulation (P < 0.001). CONCLUSION: Radial artery thrombosis is a frequent complication after transradial coronary angiography. Incidence of RA occlusion is underestimated due to the often asymptomatic clinical course. Treatment of symptomatic RA occlusion with low-molecular-weight heparins significantly increases patency rates after 4 weeks.

Telmisartan improves absolute walking distance and endothelial function in patients with peripheral artery disease.

BACKGROUND: Peripheral artery disease (PAD) is associated with high cardiovascular mortality and a poor quality of life. The AT1-receptor blocker telmisartan has been shown to have pleiotropic effects and it may also improve endothelial function. The aim of this study was to analyze the effects of telmisartan on absolute walking distance (WD) and endothelial function in patients with PAD. METHODS: In a single centre, single-blinded, prospective study, 36 patients with PAD at stage Fontaine II or higher and mild to moderate arterial hypertension were treated with telmisartan 40/80 mg once daily or placebo for 12 months. Primary endpoint was the improvement of the absolute treadmill WD. Flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT), ankle-brachial index (ABI) and disease-related quality of life (DRQL) were examined as well. RESULTS: After 12 months, maximum WD increased by 26% in the telmisartan group (P < 0.001). However, in the placebo group it was comparable to baseline. FMD rose by 40% in the telmisartan group while it deteriorated in the placebo group (P < 0.001). IMT and ABI were comparable in both groups at baseline and did not change considerably after 12 months. In non-diabetic patients (72.2%), the ABI did not change in the placebo group, whereas it increased by 11% in the telmisartan group (P < 0.001). While the DRQL remained stable in the telmisartan group, placebo treatment was associated with a marked deterioration (P < 0.01). CONCLUSION: Telmisartan improves WD and endothelial function, the ABI in non-diabetic patients and it may prevent further loss of quality of life in patients with advanced PAD.

Expanding CEP290 mutational spectrum in ciliopathies.

Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

[The efficacy of local anaesthetics administered prior to castration of male suckling piglets]. / Untersuchungen zur Wirksamkeit von Lokalanästhetika bei der Kastration von männlichen Saugferkeln.

Objective of this study was to examine the efficacy of various local anaesthetics (Procaine Hydrochloride, Procaine Hydrochloride + Epinephrin, Lidocaine Hydrochloride) used in castration of four to six day old male piglets. Animals of the control groups (I-IV) were treated analogous to the castration groups (V-IX) but not castrated. In groups I and V 15 minutes prior to castration/fixation the piglets were fixed as for injection but not medicated. The injection volume was standardised to 0.5 ml per testes. Local anaesthetics were applicated intratesticularly in five groups and intrascrotarly in two groups. Blood samples were taken shortly before and one, four and 24 hours after castration/fixation. The serum cortisol level was analysed to determine pain induced neuroendocrine stress reaction. To proof the tissue tolerance creatinkinase-(CK) and aspartataminotransferase-(AST) concentration were analysed. One, seven and 14 days after castration the healing process were evaluated by a wound score. Groups I to IV did not show any increase of the serum cortisol level. Contrary one hour after castration the average cortisol concentration of castration groups (V-IX) rose significantly. Cortisol concentration of the medicated castration groups (VI-IX) showed a comparable or even higher cortisol level as the not medicated control group (V). Determination of CK and AST indicated no tissue damage of the application of the local anaesthetics. The administration of local anaesthetics prior to the castration did not have any effect on the wound healing process. The results of this study lead to the conclusion that injection of local anaesthetics show a good tissue tolerance but the call for reduction of the castration pain by presurgical injection of Procaine Hydrochloride or Lidocaine Hydrochloride is not justified.

Right-sided diaphragmatic eventration: a rare cause of non-immune hydrops fetalis.

We report 2 cases of non-immune hydrops fetalis (NIHF) in which autopsy findings revealed an association with right-sided congenital diaphragmatic eventration (CDE). Both patients born at 30 weeks of gestation presented with severe generalized skin oedema, pleural effusions and ascites. They both died shortly after birth of cardiorespiratory insufficiency due to lung hypoplasia and low output heart failure. The right thoracic cavity was filled with the membranous but intact right diaphragm covering the herniated visceral organs including parts of the liver, small bowel and colon surrounded by voluminous ascites. In similar fashion to the situation seen in congenital diaphragmatic hernia (CDH), the displaced visceral organs led to impaired lung growth resulting in important lung hypoplasia and obstructed venous return. Extravascular liquid accumulation was probably further aggravated by hypoproteinaemia secondary to liver dysfunction resulting from the venous congestion and cardiac failure. In summary, CDE is a rare condition resulting from impaired ingrowth of muscle fibres into the diaphragm during the first trimester. Prenatal differentiation of CDE from CDH is a real challenge. The association of CDE and NIHF has not been previously described.

Pathology, natural history and treatment of abdominal aortic aneurysms.

With increasing age of the population and improvement of diagnostic tools, the incidence of abdominal aortic aneurysms (AAA) has been rising steadily. Despite an improvement in operative and interventional treatment options, AAA is the cause of death in 1-3% of men over 65 years of age in industrial countries, mostly due to rupture [1]. Therefore, routine screening for AAA by ultrasonography has been postulated in the past: a 60 year old man with an abdominal aortic diameter of less than 3 cm has a life-time risk of developing AAA close to zero. However, routine screening has not been found to be cost effective. Despite of the results of two well-designed studies, the limits of AAA qualifying the patient for surgery or intervention in contrast to conservative treatment is still a matter of debate. The present review article summarizes the current knowledge of the pathology, incidence, risks, natural course as well as symptoms and current treatment strategies of AAA on the basis of the recent literature.

Expression of nitric oxide related enzymes in coronary heart disease.

Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1-3 (synthesis of NO), arginase1 (reduction of L-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1-3 (scavengers of superoxide anions), PRTMT1-3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.

Upregulation of ID protein by growth and differentiation factor 5 (GDF5) through a smad-dependent and MAPK-independent pathway in HUVSMC.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene.

BACKGROUND: The majority of COL2A1 missense mutations are substitutions of obligatory glycine residues in the triple helical domain. Only a few non-glycine missense mutations have been reported and among these, the arginine to cysteine substitutions predominate. OBJECTIVE: To investigate in more detail the phenotype resulting from arginine to cysteine mutations in the COL2A1 gene. METHODS: The clinical and radiographic phenotype of all patients in whom an arginine to cysteine mutation in the COL2A1 gene was identified in our laboratory, was studied and correlated with the abnormal genotype. The COL2A1 genotyping involved DHPLC analysis with subsequent sequencing of the abnormal fragments. RESULTS: Six different mutations (R75C, R365C, R519C, R704C, R789C, R1076C) were found in 11 unrelated probands. Each mutation resulted in a rather constant and site-specific phenotype, but a perinatally lethal disorder was never observed. Spondyloarthropathy with normal stature and no ocular involvement were features of patients with the R75C, R519C, or R1076C mutation. Short third and/or fourth toes was a distinguishing feature of the R75C mutation and brachydactyly with enlarged finger joints a key feature of the R1076C substitution. Stickler dysplasia with brachydactyly was observed in patients with the R704C mutation. The R365C and R789C mutations resulted in classic Stickler dysplasia and spondyloepiphyseal dysplasia congenita (SEDC), respectively. CONCLUSIONS: Arginine to cysteine mutations are rather infrequent COL2A1 mutations which cause a spectrum of phenotypes including classic SEDC and Stickler dysplasia, but also some unusual entities that have not yet been recognised and described as type II collagenopathies.

Winchester syndrome caused by a homozygous mutation affecting the active site of matrix metalloproteinase 2.

The inherited osteolysis syndromes are a heterogeneous group of skeletal disorders whose classification is still uncertain. Three osteolysis syndromes show autosomal recessive inheritance and multicentric involvement: Torg syndrome (OMIM 259600), Winchester syndrome (OMIM 277950) and Nodulosis-Arthropathy-Osteolysis syndrome (NAO; OMIM 605156). The 2001 Nosology of the International Skeletal Dysplasia Society (Hall CM, Am J Med Genet 2002: 113: 65) classifies NAO as a variant of Torg syndrome, while Winchester syndrome is considered as a separate disorder. Recently, mutations in the matrix metalloproteinase 2 (MMP2) gene were identified in affected individuals with a clinical diagnosis of NAO in two Arab families. We report a homozygous missense mutation (E404K) in the active site of MMP2 in a 21-year-old woman with a severe form of osteolysis best compatible with a diagnosis of Winchester syndrome. The clinical and molecular findings suggest that Torg, NAO and Winchester syndromes are allelic disorders that form a clinical spectrum.

Fatal outcome in a female monozygotic twin with X-linked hypohydrotic ectodermal dysplasia (XLHED) due to a de novo t(X;9) translocation with probable disruption of the EDA gene.

UNLABELLED: Ectodermal dysplasias are a group of congenital disorders with defective development of the epidermis and its appendages. X-linked hypohydrotic ectodermal dysplasia (XLHED; OMIM 305100) is the most common form of ectodermal dysplasia. We report on two monozygotic twin girls with XLHED due to a t(X;9) translocation causing a disruption of the EDA gene and non random inactivation of the normal X chromosome. One of the girls died unexpectedly at 2.5 years of age. Autopsy revealed that lack of normal tracheobronchial secretions leading to complete tracheal obstruction by mucous debris was the probable cause of death. CONCLUSION: Morbidity and mortality of ectodermal dysplasias in infancy and early childhood can be significant. Early diagnosis by paediatricians is important and complications should be anticipated.

A characteristic EEG pattern in 4p-syndrome: case report and review of the literature.

UNLABELLED: Deletions on the short arm of chromosome 4 cause Wolf-Hirschhorn syndrome (WHS) and Pitt-Rogers-Danks syndrome (PRDS). WHS is associated with severe growth and mental retardation, microcephaly, a characteristic facies and congenital malformations. The PRDS phenotype is similar to WHS but generally less severe. Seizures occur in the majority of WHS and PRDS patients. Sgrò et al. [17] described a stereotypic electroclinical pattern in four unrelated WHS patients, consisting of intermittent bursts of 2-3 Hz high voltage slow waves with spike wave activity in the parietal areas during drowsiness and sleep associated with myoclonic jerks. We report a patient with PRDS and the typical EEG pattern and review 14 WHS patients with similar EEG findings reported in the literature. CONCLUSION: Awareness and recognition of the characteristic electroclinical findings in Wolf-Hirschhorn syndrome and Pitt-Rogers-Danks syndrome might help in the early diagnosis of such patients.

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.