Genetic screening of 202 individuals with congenital limb malformations and requiring reconstructive surgery.

BACKGROUND: Congenital limb malformations (CLMs) are common and present to a variety of specialties, notably plastic and orthopaedic surgeons, and clinical geneticists. The authors aimed to characterise causative mutations in an unselected cohort of patients with CLMs requiring reconstructive surgery. METHODS: 202 patients presenting with CLM were recruited. The authors obtained G-banded karyotypes and screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for point mutations using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. Multiplex ligation dependent probe amplification (MLPA) kits were developed and used to measure copy number in GLI3, HOXD13, ROR2, SALL1, SALL4, TBX5 and the ZRS of SHH. RESULTS: Within the cohort, causative genetic alterations were identified in 23 patients (11%): mutations in GLI3 (n = 5), HOXD13 (n = 5), the ZRS of SHH (n = 4), and chromosome abnormalities (n = 4) were the most common lesions found. Clinical features that predicted the discovery of a genetic cause included a bilateral malformation, positive family history, and having increasing numbers of limbs affected (all p<0.01). Additionally, specific patterns of malformation predicted mutations in specific genes. CONCLUSIONS: Based on higher mutation prevalence the authors propose that GLI3, HOXD13 and the ZRS of SHH should be prioritised for introduction into molecular genetic testing programmes for CLM. The authors have developed simple criteria that can refine the selection of patients by surgeons for referral to clinical geneticists. The cohort also represents an excellent resource to test for mutations in novel candidate genes.

Identification and characterization of multiple splice forms of the human interleukin-23 receptor alpha chain in mitogen-activated leukocytes.

The signalling of interleukin-23 (IL-23) and its receptor (IL-23R) is a key element in the differentiation of T cells to the Th17 phenotype. Here, we present the identification and characterization of human IL23R splice variants resulting from alternative splicing of the IL23R mRNA, from activated human leukocytes, following the analysis of IL23R cDNA. Twenty-four different IL23R transcripts were observed in this study, which may potentially lead to an alteration in the protein coding region of IL-23R alpha. Consequently, by analysing amino acid sequences deduced from alternatively spliced mRNA sequences, four different putative premature early termination forms of IL-23R alpha: (1) a very short 'IL-23R alpha', (2) an IL-23R alpha containing only the extracellular region, (3) a IL-23R alpha with truncated intracellular domain and (4) in-frame exon-skipping causing changes to the extracellular region of the IL-23R alpha were revealed. These changes may affect the function of IL-23R by altering the ligand-binding interaction, producing a soluble form of the receptor to act as a decoy receptor and/or modify the IL-23/IL-23R signalling, respectively. Taken together, identification of potentially functional splice variants of IL23R underscores the biological diversity of the IL23R gene and will aid in the understanding of the gene's function in normal and pathological conditions.

A novel insertion variant of the human IL-23 receptor-alpha chain transcript.

Alternative splicing of mRNA is an important mechanism for organisms to enhance protein diversity from a limited number of genes. In this report, we described a novel exon insertion in the interleukin 23 (IL-23) receptor between exons 9 and 10, denoted as exon 9a. This 162 base-pair insertion was the only insertion variant discovered in more than 20 IL23R deletion variants found in the mRNA of mitogen-stimulated peripheral blood mononuclear cells (PBMC). Sequence analysis revealed that a pair of GT/AG splice donor-acceptor site and several putative cis-acting sequences were present; the insertion was identified throughout the genome and found to contain homology to the L1 retrotransposon protein. This report describes an insertion in the IL-23 receptor and due to consequent early termination within the intracellular region, causing a possible non-responsive receptor isoform.

The major immunogenic epitopes of Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a protein expressed consistently in EBV-infected cells and EBV-associated malignant tissues. A panel of monoclonal antibodies (MAbs) was generated against the C terminus of EBNA-1 and evaluated for the detection of EBNA-1 in different cell lines. The epitopes recognized were mapped. Since sequence variations of EBNA-1 have been reported in nasopharyngeal carcinoma (NPC) tissues and in infected healthy individuals, the ability of these MAbs to recognize a recombinant protein derived from an NPC biopsy was also analysed. MAb 4H11 appeared to react with EBNA-1 sequences from different sources, whereas MAbs 5C11, 5F12 and 8F6 failed to recognize a recombinant EBNA-1 protein cloned from an NPC patient. Using different recombinant EBNA-1 fragments in an immunoblot format, this study demonstrates that the domain bounded by amino acids 408 and 498 is very immunogenic in mice in that epitopes in this region are recognized by various MAbs. Amino acid sequences of EBNA-1 were also deduced from nucleotide sequences amplified from three Burkitt's lymphoma cell lines, two spontaneous lymphoblastoid cell lines, two NPC biopsies and one NPC hybrid cell line, NPC-KT, and compared to the sequence from B95-8. The amino acid sequence of EBNA-1 in Akata is almost identical to that in an NPC biopsy, except for amino acid 585. The results of this study indicate that the immunogenic epitopes of EBNA-1 are highly variable.

Epidemiology of vertebral fractures in women.

Vertebral fracture prevalence was assessed in an age-stratified random sample of Rochester, Minnesota women aged 50 years and over. Vertebral fractures, including wedge and concavity as well as compression fractures, were common and increased with age. The estimated incidence of new vertebral fractures also rose with age, reaching 29.6 per 1,000 person-years in women aged greater than or equal to 85 years. The prevalence of one or more vertebral fractures also increased with declining bone mass, reaching 42% in women with spinal bone mineral density less than 0.6 g/cm2 by dual photon absorptiometry. Bone mass and age contributed independently to the risk of vertebral fracture, but "age" may reflect other manifestations of osteoporosis.

Lifetime fracture risk: an approach to hip fracture risk assessment based on bone mineral density and age.

Hip fractures occur late in life following a substantial reduction in skeletal mass. If risk for such fractures could be predicted early, efforts to prevent excessive bone loss would be more successful and could be directed at the individuals most likely to be affected. With this objective in mind, we devised an approach to estimating the lifetime risk of a proximal femur fracture based on age and on current femoral bone mineral density, using population-based data from ongoing studies of osteoporosis and fractures among Rochester, Minnesota, women. Our calculations indicate that, at any given age, the lifetime risk of a proximal femur fracture rises as current bone density diminishes. At any given level of femoral bone density, lifetime risk rises with younger age and increasing life expectancy. While these trends seem robust, estimates of risk vary substantially with the assumptions that underlie the model. Consequently, these assumptions must be validated before our findings can be applied clinically to predict risk for individual patients.

Errors in longitudinal measurements of bone mineral: effect of source strength in single and dual photon absorptiometry.

The effect of changing strength during the useful life of a radiation source was evaluated in studies performed on four dual photon (DPA) and two single photon (SPA) bone absorptiometry instruments. Two DPA units and one SPA unit did not show any systematic dependence of measured bone mineral content or bone mineral areal density (BMD) on source activity when evaluated over an entire source life. One DPA and one SPA instrument, however, showed significant time trends associated with source activity. The fourth DPA instrument had a significant linear decrease in BMD over a source life in the automatic mode but performed better in the manual mode.

Risk of osteoporotic fractures in women with breast cancer: a population-based cohort study.

A population-based study was undertaken to assess the risk of osteoporotic fractures in an inception cohort of breast cancer patients and an age-matched cohort of women from the community. Prior to the index date, 9.4% of cases and 12.3% of controls (p = 0.30) had one or more osteoporotic fractures. After the index date, these proportions were 16.2 and 20.0% (p = 0.28), but follow-up was shorter for cases due to reduced survival. When duration of follow-up was accounted for in a person-years analysis, the relative risk of any fracture was 1.0 and for any osteoporotic fracture was 0.9 (95% C.I. 0.7-1.2). After adjusting for other factors in a proportional hazards model, the relative risk of any osteoporotic fracture in breast cancer cases compared to controls was 0.9. Despite contentions that breast cancer patients have more often been exposed to estrogenic factors, we found little to suggest that such women are substantially protected from osteoporotic fractures.