Use of the Contralateral Glenoid for Calculation of Glenoid Bone Loss: A Cadaveric Anthropometric Study.

PURPOSE: The purpose of this study was to determine if there are significant side-to-side anthropometric differences between paired glenoids. METHODS: Forty-six matched-pair cadaver glenoids were harvested, and their glenoid heights (GHs) and glenoid widths (GWs) were measured with digital calipers. The glenoid surface area was calculated using the standard assumption that the inferior two-thirds of the glenoid is a perfect circle. RESULTS: There was a statistically significant difference between matched-pair GHs of 0.96 ± 3.07 mm (P = .020) and GWs of 0.46 ± 1.64 mm (P = .033). There was a significant difference of glenoid cavity area of 20.30 ± 81.53 mm2 (P = .044), or a difference of ∼3%. A total of 4 of 46 pairs of glenoids (8.6%) showed a difference in width >3 mm. CONCLUSIONS: This study demonstrates the fallacy of use of the contralateral glenoid in measuring glenoid bone loss. Although many paired samples exhibited similar side-to-side glenoid measurements, the number of cadaveric pairs that showed differences of >3 mm was substantial. Caution should be taken when using calculation methods that include this assumption for surgical decision making, as surface area, GW, and GH were all shown to have statistically significant side-to-side differences in their measurements. CLINICAL RELEVANCE: Many methods exist for measuring glenoid bone loss after anterior shoulder dislocation, but some of the current methods may be inaccurate and lead to unreliable estimations.

DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics.

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6-mutation negative individuals with non-syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations.

Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments.

Positional cloning of the gene associated with X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis(RS) is a recessively inherited vitreo-retinal degeneration characterized by macular pathology and intraretinal splitting of the retina. The RS gene has been localized to Xp22.2 to an approximately 1 Mb interval between DXS418 and DXS999/DXS7161. Mapping and expression analysis of expressed sequence tags have identified a novel transcript, designated XLRS1, within the centromeric RS locus that is exclusively expressed in retina. The predicted XLRS1 protein contains a highly conserved motif implicated in cell-cell interaction and thus may be active in cell adhesion processes during retinal development. Mutational analyses of XLRS1 in affected individuals from nine unrelated RS families revealed one nonsense, one frameshift, one splice acceptor and six missense mutations segregating with the disease phenotype in the respective families. These data provide strong evidence that the XLRS1 gene, when mutated, causes RS.

Mutation screening of the RYR1-cDNA from peripheral B-lymphocytes in 15 Swedish malignant hyperthermia index cases.

BACKGROUND: Malignant hyperthermia (MH), linked to the ryanodine receptor 1 gene (RYR1) on chromosome 19, is a potentially lethal pharmacogenetic disorder which may lead to a disturbance of intracellular calcium homeostasis when susceptible individuals are exposed to halogenated anaesthetics, suxamethonium, or both. Central core disease (CCD) is a rare dominantly inherited congenital myopathy allelic to MH-susceptibility. METHODS: In this study, 14 unrelated MH-susceptible probands and one CCD patient from Sweden were screened for mutations in the RYR1. Since the RYR1 is also expressed in B-lymphocytes, RYR1-cDNA was transcribed from total RNA extracted from white blood cells. RESULTS: We detected two known RYR1 mutations and two previously described unclassified sequence variants. In addition, six novel sequence variants were detected. All mutations or sequence variants were verified on genomic DNA. Seven of the probands did not show any candidate mutation, although the total coding region of RYR1 was sequenced. Segregation data in in vitro contracture tested family members of three probands support a causative role of three of the novel sequence variants. CONCLUSIONS: Our study contributes to the genetic aetiology of MH in Sweden, but also raises questions about the involvement of genes other than RYR1 since nearly half of the probands did not show any sequence variants in the total coding region of the RYR1.

The role of caspases in photoreceptor cell death of the retinoschisin-deficient mouse.

Early schisis cavities in the retinal bipolar cell layer accompanied by progressive loss of cone and rod photoreceptor cells are the hallmark of the retinoschisin-deficient (Rs1h(-/Y)) murine retina. With this study we aimed at elucidating the molecular events underlying the photoreceptor cell death in this established murine model of X-linked juvenile retinoschisis. We show that photoreceptor degeneration in the Rs1h(-/Y) mouse is due to apoptotic events peaking around postnatal day 18. Cell death is accompanied by increased expression of initiator and inflammatory caspases but not by downstream effector caspases. The strong induction of caspase-1 (Casp1) prompted us to explore its involvement in the apoptotic process. We therefore generated double knock-out mice deficient for both retinoschisin and Casp1. No direct influence of the Casp1 genotype on apoptosis could be identified although striking differences in the overall number of resident microglia were observed independent of the Rs1h genotype.

[Stability of voriconazole in infusion bags]. / Stabilität von Voriconazol-Konzentrat und Voriconazol-Infusionsbeuteln.

Stability of voriconazole in infusion bags The dosage of i.v. administered voriconazole in adults and children is body weight based. For this reason an individual preparation is necessary in those patients. Further stability data were needed for a central production in the hospital pharmacy department, however. Three concentrates and three bags were produced (day 1) according to the procedures given by the product leaflet. Our data shows that the ready to use bag and the concentrate solution is stable at 2-8 degrees C for 32 days.

Mutations in the VMD2 gene are associated with juvenile-onset vitelliform macular dystrophy (Best disease) and adult vitelliform macular dystrophy but not age-related macular degeneration.

Recently, the VMD2 gene has been identified as the causative gene in juvenile-onset vitelliform macular dystrophy (Best disease), a central retinopathy primarily characterised by an impaired function of the retinal pigment epithelium. In this study we have further characterised the spectrum of VMD2 mutations in a series of 41 unrelated Best disease patients. Furthermore we expanded our analysis to include 32 unrelated patients with adult vitelliform macular dystrophy (AVMD) and 200 patients with age-related macular degeneration (AMD). Both AVMD and AMD share some phenotypic features with Best disease such as abnormal subretinal accumulation of lipofuscin material, progressive geographic atrophy and choroidal neovascularisation, and may be the consequence of a common pathogenic mechanism. In total, we have identified 23 distinct disease-associated mutations in Best disease and four different mutations in AVMD. Two of the mutations found in the AVMD patients were also seen in Best disease suggesting a considerable overlap in the aetiology of these two disorders. There were no mutations found in the AMD group. In addition, four frequent intragenic polymorphisms did not reveal allelic association of the VMD2 locus with AMD. These data exclude a direct role of VMD2 in the predisposition to AMD.

Identifying differentially expressed genes in the mammalian retina and the retinal pigment epithelium by suppression subtractive hybridization.

Retina and retinal pigment epithelium (RPE) cells are of neuroectodermal origin with highly specialized functions in light perception. Identification and characterization of genes differentially expressed in these cells will greatly aid our understanding of their functional roles in retinal biology. As a source enriched for gene transcripts from the retina/RPE, we generated a human retina and a bovine RPE cDNA library applying the PCR-based technique of suppression subtractive hybridization (SSH). Sequencing of 1,080 retina and 2,350 RPE SSH clones resulted in the identification of 321 and 343 non-redundant human transcripts, respectively. Of these, only 27 genes were in common between the two cDNA libraries. One transcript expressed exclusively in retina and RPE is the novel gene C4orf11 which is comprised of four exons on chromosome 4q21.2. We report the full-length cloning of two isoforms of C4orf11, 919 bp and 857 bp in length, both of which contain four identical open reading frames (ORFs). While ORFs 1 to 3 show no homologies to known proteins or protein domains, ORF4 reveals 50% sequence identity to RPE-spondin, a hypothetical protein on 8q13.3 with unknown function. We demonstrate that both the retina and the RPE SSH cDNA libraries are excellent resources for identifying known and novel genes exclusively or abundantly expressed in the retina/RPE complex. In combination with other approaches such as microarray analysis or serial analysis of gene expression (SAGE), the availability of highly sensitive and specific SSH cDNA libraries will facilitate the comprehensive description of the retina/RPE transcriptome.

PTEN mutations do not cause nuclear beta-catenin accumulation in endometrial carcinomas.

PTEN: and beta-catenin mutations constitute the predominant genetic alterations in endometrioid carcinomas of the endometrium. PTEN encodes a dual-specificity phosphatase with lipid phosphatase and protein tyrosine phosphatase activities that regulate both apoptosis and interactions with the extracellular matrix. Recent studies have associated PTEN mutations with tumorigenesis of prostate carcinoma via the Wnt signaling pathway, leading to nuclear beta-catenin accumulation. To elucidate the potential interaction of PTEN and beta-catenin in endometrial cancer, we performed mutation analyses of the entire PTEN gene and of exon 3 of the beta-catenin gene that is most frequently targeted by mutations. A total of 82 endometrial carcinomas comprising 62 type I endometrioid carcinomas and 20 type II high-grade carcinomas were investigated. In addition in a subset of 22 carcinomas, the intracellular beta-catenin distribution was analyzed by immunohistochemistry. Overall, 20 (24.4%) of 82 tumors revealed mutations in the PTEN gene, and 16 (19.5%) of 82, in the beta-catenin gene. Six tumors (7.3%) showed mutations in both the PTEN and beta-catenin gene. Mutations were mainly detected in endometrioid carcinomas of the endometrium. As expected, a striking nuclear accumulation of beta-catenin could be shown in tumors with beta-catenin mutations. In the vast majority of tumors with PTEN mutations, a regular staining pattern of the cytoplasmic and membranous compartments was found. We therefore conclude that, in contrast to prostate cancer, mutations in the PTEN gene seem not to affect cellular distribution of the beta-catenin protein in endometrial carcinomas.

Chondrogenesis of limb-bud cells: improved culture method and the effect of the potent teratogen retinoic acid.

A new apparatus for the mild and reproducible dissociation of rat limb buds into single cells is described. The growth and differentiation of the embryonic mesenchyme cells into chrondrocytes was markedly affected by the conditions of tissue dissociation and the serum used. The degree of chondrogenesis was assessed by alcian blue staining, specific for cartilage matrix. Retinoic acid, a known teratogen, inhibited chondrogenesis completely in a dose-dependent manner. The determination of the quantifiable end-point, IC50 in this cell-culture system, may be suitable to compare the activity of various retinoids and eventually to determine their teratogenic potential.

EST mining of the UniGene dataset to identify retina-specific genes.

Age-related macular degeneration (AMD) is a multifactorial disorder affecting the visual system with a high prevalence among the elderly population but with no effective therapy available at present. To better understand the pathogenesis of this disorder, the identification of the genetic factors and the determination of their contribution to AMD is needed. Towards this goal, we are pursuing a strategy that makes use of the EST data processed in the UniGene database and aims at the generation of a comprehensive catalogue of genes preferentially active in the human retina. Subsequently, these genes will be systematically assessed in AMD. We performed a retina EST sampling and obtained a total of 673 clusters containing only retina ESTs as well as 568 clusters with at least 30% of the ESTs in each cluster originating from retina cDNA libraries. Of these, 180 representative EST clusters with varying retina and non-retina EST contents were analyzed for their in vitro expression. This approach identified 39 transcripts with retina-specific expression. One of these genes (C18orf2) mapping to chromosome 18 was further characterized. Multiple C18orf2 transcripts display a complex pattern of differential splicing in the human retina. The various isoforms encode hypothetical polypeptides with no homologies to known proteins or protein motifs.

Molecular defects in alkaptonuria.

At the dawn of human genetics Sir Archibald Garrod used alkaptonuria as a paradigm to demonstrate the applicability of the Mendelian laws to men and to develop the concept of inborn errors of metabolism. The human cDNA for homogentisate 1,2 dioxygenase was identified due to its homology to the corresponding mouse enzyme and was screened for mutations in alkaptonuric patients from Slovakia. Homozygous mutations were found in four unrelated families and their segregation with the disease was demonstrated. One of the mutations, observed in two families, leads to a frame-shift and thus is unlikely to produce functional protein. The data formally establish the homogentisate 1,2 dioxygenase gene (HGD) as the molecular cause of alkaptonuria and allow for the development of molecular carrier tests in populations at risk.

[Venous cross-sectional area: measured or calculated?]. / Venenquerschnittsfläche: messen oder berechnen?

AIM: The aim of this study was to compare measured versus calculated venous cross-sectional area in healthy subjects in a standing and a lying position with normal breath-ing and during Valsalva manoeuvre. METHOD: Measurements were carried out in 30 venous segments (the common femoral vein CFV, the superficial femoral vein SFV, the greater saphenous vein GSV) of 5 healthy volunteers (4 female, 1 male) with a median age of 28.7 years (range 23.4-46.7 years) in supine and standing position, while normally breathing and during a standardised Valsalva manoeuvre. Venous diameters were measured from B-mode in longitudinal view while cross-sectional areas were planimetrically assessed from transverse B-mode as recorded on video (S-VHS). The mathematical calculation of areas followed the formula (0.5 diameter)2 x pi. All investigations were performed 3 times; mean values from these 3 measurements were used for further computation. Measurements were performed using the NIH Image 1.6 program. RESULTS: Correlation coefficients r of the calculated versus the measured venous area while normal breathing in standing and in lying subjects were: 0.92 and 0.82 in the CFV, 0.92 and 0.84 in the SFV as well as 0.98 and 0.97 in the GSV, respectively. During Valsalva manoeuvre in standing and lying subjects the correlation coefficients r amounted to: 0.94 and 0.93 in the CFV, 0.92 and 0.94 in the SFV as well as 0.99 and 0.98 in the GSV, respectively. CONCLUSIONS: In healthy volunteers measured and calculated venous cross-sectional area at rest and during Valsalva manoeuvre correlate well. Correlation is numerically better in standing compared to lying subjects while normal breathing. Calculated venous area is accurate and can be used for further calculations.

The value of rapid D-dimer testing combined with structured clinical evaluation for the diagnosis of deep vein thrombosis.

PURPOSE: Large studies have shown that most cases referred for duplex sonography for suspected deep vein thrombosis (DVT) have normal scan results. For medical and economic reasons, a preselection procedure, which allows the detection of true-negative cases before duplex scanning, is required; this procedure should be characterized by a high sensitivity and a high negative predictive value. METHODS: In 343 patients (398 lower extremities) with suspected DVT, the DVT probability was clinically assessed, and a whole blood D-dimer agglutination test and a duplex scan were performed. The diagnostic sensitivities of the D-dimer test alone, a high clinical DVT probability alone, and the combination of both were evaluated. RESULTS: The sensitivity values for the D-dimer test to diagnose proximal and distal DVTs were 88.7% and 80.9%, the negative predictive values (NPV) were 96.3% and 97.9%, and the specificity and the positive predictive value (PPV) were 54.8% and 49.6% and 26.6% and 8.2%, respectively. The sensitivities of the clinical DVT probability assessment for the diagnosis of proximal and distal DVTs were 83.9% and 66.7%, respectively; the corresponding NPVs were 94.9% and 96.5%, respectively. The specificity was 56.1% and 50.8%, and the PPVs were 26.1% and 7.0%, respectively. The combined use of the results of the clinical probability assessment and the D-dimer test resulted in sensitivities for proximal and distal DVTs of 98.4% and 90.5%, NPVs of 99.3% and 98.6%, a specificity of 43.4% and 38.4%, and PPVs of 24. 3% and 7.6%, respectively. CONCLUSION: The combined use of a clinical DVT probability assessment scheme and the D-dimer test largely avoids false negative results, has a high sensitivity and NPV, helps to reduce the costs of DVT diagnosis, and may, in the future, be useful as a preselection procedure before duplex sonography.

Isolation and characterization of the murine X-linked juvenile retinoschisis (Rs1h) gene.

X-linked juvenile retinoschisis (RS) is a vitreoretinal degeneration affecting only males. Recently, the RS1 gene underlying this common cause of early vision loss was identified and shown to encode a 224-amino acid precursor protein including a 23-residue leader sequence as well as a highly conserved discoidin motif at the C-terminus. Functional studies in other proteins with discoidin motifs have implicated this domain in phospholipid binding and cell-cell interactions on membrane surfaces. Thus, similar functional properties may exist for RS1 and may be related to the histopathological findings in RS. In order to further pursue the pathophysiology of RS and to understand RS1 function in early eye development, we now report the identification and characterization of the complete murine Rs1h gene. The full-length Rs1h cDNA was isolated by RT-PCR with degenerate oligonucleotide primers designed from human RS1 cDNA sequences. Subsequently, the exon/intron structure was determined in genomic DNA from mouse strain 129/SvJ. We show that human and murine RS1 coding sequences, exon/intron boundaries, as well as retina-specific expression, are highly conserved between the two species. The conceptual human and murine protein sequences reveal 96% amino acid identity with no amino acid changes within the discoidin domain. In addition, alignment of 5'-flanking sequences upstream of the human and mouse RS1 translation initiation sites identified putative binding sites for several transcription factors including CRX, a homeodomain transcription factor known to activate the transcription of several photoreceptor-specific genes.

Assessment of RS1 in X-linked juvenile retinoschisis and sporadic senile retinoschisis.

The RS1 gene is the causative gene in X-linked juvenile retinoschisis (RS). We have screened this gene for mutations in 13 patients with RS and in 7 probands with senile retinoschisis, a sporadic, later-onset form of retinoschisis. Mutations were detected in all RS patients. Of the 11 different mutations identified, six have been reported previously and live are novel. We did not find mutations in any of the senile retinoschisis patients and conclude that senile retinoschisis is not the result of germline mutations in the RS1 gene.

Cloning of the homogentisate 1,2-dioxygenase gene, the key enzyme of alkaptonuria in mouse.

We determined 48 amino acid residues from five peptides from the homogeneous monomer of homogentisate 1,2-dioxygenase (HGO; E.C. 1.13. 11.15) of mouse liver. After digestion with trypsin, peptides were separated by reversed phase chromatography and amino acid sequenced. The deduced codon sequence of three peptides was used to derive degenerated oligomeres. By combining these oligos, we were able to amplify fragments from 100 to 300 bases (b) from mouse liver cDNA by polymerase chain reaction after reverse transcription (RT-PCR). A fragment of 200 b was cloned and used as a probe to screen a mouse liver cDNA library. One clone from this library contained the complete cDNA-insert for HGO as determined by sequencing. The cDNA encodes for a protein of 50 kDa, as predicted. The cDNA of mouse HGO has an overall identity of 41% to the corresponding gene hmgA from Aspergillus. Sequence similarities to human expressed sequence tags (EST) clones ranged from 70% to 20%. The positions of 122 conserved amino acids could be determined by multiple sequence alignment. We identified one first intron of 928 b in the mouse gene. The gene for HGO seems to be expressed in various tissues, as shown by RT-PCR on different cDNAs. FISH experiments with the whole murine cDNA as probe clearly revealed signals at the human chromosomal band 3q13. 3-q21. This corresponds well to the previous assignment of the locus for the human alkaptonuria gene (AKU) to the same chromosomal region by multipoint linkage analysis. We therefore conclude that the HGO cDNA encodes the gene responsible for alkaptonuria.

Transcript map of a 900-kb genomic region in Xp22.1-p22.2: identification of 12 novel genes.

The Xp22.1-p22.2 interval is a focus of interest as a number of hereditary disease loci have been mapped to this region, including X-linked nonsyndromic sensorineural deafness (DFN6), X-linked juvenile retinoschisis (RS), and several X-linked mental retardation syndromes. In the course of cloning the RS gene we have assembled YAC and PAC contigs of the 900-kb candidate region delimited by DXS418 and DXS999. In this study, we now report the construction of a first transcript map of this chromosomal interval by combining exon trapping, EST mapping, and computational gene identification methods. Overall, this strategy has led to the assembly of at least 12 novel transcripts positioned within the DXS418-DXS999 region, one of these encoding a putative protein kinase motif with significant homology to the rat p58/GTA protein kinase domain and another a putative neuronal protein with strong homology to a Drosophila transcriptional repressor.

Genomic organization and chromosomal localization of the interphotoreceptor matrix proteoglycan-1 (IMPG1) gene: a candidate for 6q-linked retinopathies.

The interphotoreceptor matrix is a unique extracellular matrix occupying the space between the photoreceptors and the retinal pigment epithelium. Due to its putative function in the maintenance and integrity of the photoreceptor cells, it is conceivable that it is involved in retinal degeneration processes. More recently, a novel gene encoding a 150-kDa interphotoreceptor matrix proteoglycan, designated IMPG1, was cloned and shown to be expressed in both rod and cone photoreceptor cells. To assess this gene in human retinal dystrophies, we have now determined the genomic organization and chromosome location of IMPG1. It is composed of 17 exons ranging from 21 to 533 bp, including an alternatively spliced exon 2. Using somatic cell hybrid mapping and FISH analysis, we have assigned the IMPG1 locus to 6q13-->q15. As this interval overlaps with the chromosomal loci of several human retinopathies, including autosomal dominant Stargardt-like macular dystrophy (STGD3), progressive bifocal chorioretinal atrophy (PBCRA), and North Carolina macular dystrophy (MCDR1), IMPG1 represents an attractive candidate for these 6q-linked disorders.