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Biomedical subjects

V J Hyland

Publications and source records attributed to V J Hyland.

At least 19 recordsLinked to original sources
PubMed2003

Familial growth and skeletal features associated with SHOX haploinsufficiency.

This study was designed to determine the intrafamilial effect of SHOX haploinsufficiency on stature, by comparing the growth and phenotype of 26 SHOX haploinsufficient individuals with 45 relatives and population standards. It confirmed that SHOX haploinsufficiency leads to growth restriction from birth to final height. Compared to unaffected siblings, the SHOX haploinsufficient cohort was 2.14 SDS (3.8 cm) shorter at birth and 2.1 SDS shorter through childhood. At final height females were 2.4 SDS (14.4 cm) shorter and males 0.8 SDS (5.3 cm) shorter than normal siblings. The family height analysis suggests that the effect of SHOX haploinsufficiency on growth may have been previously underestimated at birth and overestimated in males at final height. SHOX haploinsufficiency leads to short arms in 92%, bilateral Madelung deformity in 73% and short stature in 54%. Females were more severely affected than males. We conclude that SHOX is a major growth gene and that mutations are associated with a broad range of phenotype.

Adolescent↗
PubMed2003

Effect of 24 months of recombinant growth hormone on height and body proportions in SHOX haploinsufficiency.

Leri-Weill syndrome (LWS) is a skeletal dysplasia with mesomelic short stature, bilateral Madelung deformity (BMD) and SHOX (short stature homeobox-containing gene) haploinsufficiency. The effect of 24 months of recombinant human growth hormone (rhGH) therapy on the stature and BMD of two females with SHOX haploinsufficiency (demonstrated by fluorescence in situ hybridisation) and LWS was evaluated. Both patients demonstrated an increase in height standard deviation score (SDS) and height velocity SDS over the 24 months of therapy. Patient 1 demonstrated a relative increase in arm-span and upper segment measurements with rhGH while patient 2 demonstrated a relative increase in lower limb length. There was appropriate advancement of bone age, no adverse events and no significant deterioration in BMD. In this study, 24 months of rhGH was a safe and effective therapy for the disproportionate short stature of SHOX haploinsufficiency, with no clinical deterioration of BMD.

Adolescent↗
PubMed2001

Histopathological analysis of Leri-Weill dyschondrosteosis: disordered growth plate.

Leri-Weill syndrome (LWS) is a dominant (pseudoautosomal) skeletal dysplasia with mesomelic short stature and bilateral Madelung deformity, due to dyschondrosteosis of the distal radius. It results from the loss of one copy of the Short Stature Homeobox Gene (SHOX) from the tip of the short arm of the X or Y chromosome. SHOX molecular testing enabled us to evaluate the histopathology of the radial physis in LWS patients with a documented SHOX abnormality. A widespread disorganisation of physeal anatomy was revealed with disruption of the normal parallel columnar arrangement of chondrocytes. Tandem stacking of maturing chondrocytes within columns was replaced by a side-by-side arrangement. The presence of hypertrophic osteoid with micro-enchondromata in the radial metaphysis suggests abnormal endochondral ossification. The Vickers' ligament was confirmed to blend with the triangular fibrocartilage complex (TFCC). This histopathological study demonstrates that the zone of dyschondrosteosis in LWS is characterised by marked disruption of normal physeal chondrocyte processes and that a generalised physeal abnormality is present.

Adolescent↗
PubMed2001

Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes.

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.

Adult↗
PubMed2001

Solid-phase amplification for detection of C282y and H63D hemochromatosis (HFE) gene mutations.

BACKGROUND: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. METHODS: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. RESULTS: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. CONCLUSIONS: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

Electrophoresis, Agar Gel↗
PubMed2000

Clinical findings in a patient with FGFR1 P252R mutation and comparison with the literature.

We report on a patient with the skeletal findings of Jackson-Weiss syndrome, who manifests only mild craniofacial anomalies. Molecular analysis of her fibroblast growth factor receptor 1 gene (FGFR1) identified a heterozygous P252R missense mutation, previously only reported with FGFR1-Pfeiffer syndrome like manifestations. Mutations in the immunoglobulin-like, II-III (IgII-III) linker region of FGFR1 and FGFR3 molecules may present as a skeletal dysplasia affecting the appendicular skeleton including, brachydactyly, short broad middle phalanges, phalangeal epiphyseal coning and broad halluces. This communication is a further example of the phenomenon of an activated FGFR molecule resulting in overlapping manifestations in FGFR syndromes.

Abnormalities, Multiple↗
PubMed1999

Molecular detection of prostate cells in ejaculate and urethral washings in men with suspected prostate cancer.

PURPOSE: To determine whether prostatic cells were normally present in ejaculate and if the sensitivity and specificity of the detection of malignant prostate cells in ejaculate and urethral washings from men with suspected prostate cancer could be improved using the more sensitive molecular technique of reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: RT-PCR for prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM) and Apoliprotein D (3 putative prostate-specific and/or cancer-specific markers) was performed on RNA extracts of ejaculate (80) and urethral washings (52) from 77 men with suspected prostate cancer and 12 young controls (<30 years of age) and urines from 5 men who had radical prostatectomies and 10 women. RESULTS: PSA, PSM and Apolipoprotein D expression was detected in ejaculates and urethral washings from both patient and control groups. No differences were observed in the results obtained for 58 men with suspected or 19 men with confirmed prostate cancer or the 18 vasectomized men within the patient group. Urines from the 5 men who had radical prostatectomies and 10 women were all negative for PSA, but PSM was detected in 2 female urines and in 3 radical prostatectomy samples. As few as 10 LNCaP prostate tumor cells could be detected by PSA RT-PCR when added to female urine. CONCLUSION: We have established a sensitive method of detecting prostatic cells in ejaculate and urethral washings and shown that PSA RT-PCR is a reliable indicator of prostate cells in these samples. However, RT-PCR for PSA, PSM and Apoliprotein D were not useful for discriminating malignant from non-malignant prostate cells.

Adult↗
PubMed1999

Glucocorticoids differentially inhibit expression of the RET proto-oncogene.

The RET proto-oncogene encodes a receptor tyrosine kinase activated by the binding of factors from the glial cell line-derived neurotrophic factor (GDNF) family to receptor-alpha components such as GDNF family receptor alpha-1 (GFR alpha-1). Mutations within the sequence of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2), an inherited tumor syndrome characterized by the development of medullary thyroid carcinoma (MTC) and other neuroendocrine tumors. Despite Northern analysis showing that RET is expressed in the majority of MTCs, the factors regulating this expression are poorly understood. To address this issue we examined RET expression in response to glucocorticoids in the TT cell line, derived from a metastatic MTC. The synthetic glucocorticoid dexamethasone was found to reduce RET expression at both mRNA and protein levels. This effect was dose responsive and maximal at 24 h. The reduction in RET mRNA was shown to be specific to glucocorticoids and was also seen in a primary MTC culture. Nuclear run-on studies revealed the reduction in steady-state RNA to be due to a decrease in RET mRNA transcription and the effect was shown to be independent of new protein synthesis or RNA stability. Dexamethasone was also found to exert an inhibitory effect upon cell growth, suggesting a potential use for glucocorticoids in the treatment of medullary carcinoma and MEN 2.

Carcinoma, Medullary↗
PubMed1998

Absence of MEN2A- or 2B-type RET mutations in primary neuroblastoma tumour tissue.

Specific germline mutations in the RET proto-oncogene predispose to the familial cancer syndromes: multiple endocrine neoplasia (MEN) types 2A and 2B, and familial medullary thyroid carcinoma. Expression of the RET receptor tyrosine kinase is tightly restricted to tumours of neural crest origin, such as neuroblastoma, and neuroblastoma has been observed in RET transgenic mice. Neuroblastoma tumour cell lines transfected with the MEN2A RET gene exhibit spontaneous neuritic differentiation, whereas MEN2B-type RET transfectants demonstrate altered cell adhesion and enhanced metastatic potential. In this study, the authors examined genomic DNA from 26 primary neuroblastoma tumours for MEN2A and MEN2B RET mutations, using restriction enzyme digestion of polymerase chain reaction products as an alternative to direct sequencing. Examination of RET exons 10 (codons 611, 618, 620), 11 (codons 632, 633, 634) and 16 (codon 918) in all 26 tumours revealed no RET mutations. Taken together these data suggest that abnormalities of the RET signalling pathway, rather than oncogenic, MEN2-type RET activation by mutation, may play a role in neuroblastoma tumorigenesis.

DNA, Neoplasm↗
PubMed1996

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro.

OBJECTIVE: To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet-endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovas- cular human umbilical vein endothelial cells (HUVE). METHODS: Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor alpha (TNF alpha). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS: SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF alpha treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E- selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF alpha treatment and then declined to near basal levels by 24-48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF alpha stimulated endothelial cells correlated with their respective messenger RNA levels. CONCLUSION: Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.

Agglutinins↗
PubMed1996

The identification of false positive responses to the pentagastrin stimulation test in RET mutation negative members of MEN 2A families.

OBJECTIVE: The pentagastrin stimulation test is the traditional test used for the identification of asymptomatic individuals in multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). The identification of mutations in the RET proto-oncogene segregating with the disease phenotype in MEN 2A and FMTC families has made it possible to re-examine the validity of using this test for the identification of affected family members. DESIGN: Sequential and single pentagastrin stimulation test data were collected following the identification of RET mutation positive and RET mutation negative members of families with MEN 2A or FMTC. PATIENTS: RET mutations were identified in 16 Australian and New Zealand MEN 2A or FMTC families. An analysis of 39 individuals from these families was included in this study. Thirty-two individuals (14 males, 18 females) had previously been determined as RET mutation negative. Seven individuals (6 males, 1 female) had previously been determined as RET mutation positive. Two RET mutation negative males had thyroidectomy based on prior pentagastrin test results. MEASUREMENTS: Serum calcitonin levels in response to stimulation with pentagastrin were measured at 0, 1, 2, 5 and 10 minutes post injection. Mutation analysis of the RET proto-oncogene was performed in all individuals. In two RET mutation negative individuals from two MEN 2A families, thyroidectomy was performed and C-cells were quantitated in order to determine the diagnosis of C-cell hyperplasia. RESULTS: There was a statistically significant difference (P < 0.013) between RET mutation negative male and female mean peak calcitonin responses of 282 +/- 236 and 96 +/- 62 (mean +/- SD) ng/l respectively. False positive responses to pentagastrin stimulation were identified in seven individuals who were RET mutation negative in two of the 16 families. Histologic examination of the thyroid glands in the two RET mutation negative individuals who had thyroidectomy demonstrated C-cell hyperplasia in one but not in the other. CONCLUSIONS: There is considerable overlap between pentagastrin test results in individuals who are RET mutation positive and those who are RET mutation negative. These results indicate a need for routine performance of RET proto-oncogene analysis on all individuals at risk of developing MEN 2A or FMTC and a coupling of pentagastrin test results and RET proto-oncogene analysis in the decision to proceed with thyroidectomy.

Adolescent↗
PubMed1995

Cytokine production by normal human monocytes: inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1Ra) gene polymorphism.

Monocytes from different individuals show variable cytokine production in response to a variety of stimuli. We wished to determine the sets of conditions (cytokine combinations) that would enable us to demonstrate stable inter-individual differences in the production of IL-1 alpha, IL-1 beta, IL-1Ra, on-6 and tumour necrosis factor-alpha (TNF-alpha) by monocytes. We assessed the ability of a number of recombinant human cytokines (granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), TNF-alpha, IL-4, IL-6, transforming growth factor-beta (TGF-beta), IL-10 and IL-1Ra)) to stimulate or inhibit the production of one or more of these monocyte products. GM-CSF was found to stimulate the production of all five of these cytokines in a highly reproducible manner. TNF-alpha also up-regulated production of IL-1 alpha, IL-1 beta, IL-1Ra and IL-6 by monocytes, but the variability in the results of cells cultured from the same individuals on different occasions was greater. Other cytokines either stimulated production of only some of the five cytokine products tested, or stimulated the production of some cytokine products while inhibiting production of others. This was especially evident when cytokines were used in combination with GM-CSF: IFN-gamma down-regulated production of IL-1Ra while up-regulating the production of IL-1 alpha/beta, IL-6 and TNF-alpha, while IL-4 had the exact opposite effect. Polymorphisms in regions of cytokine genes that affect transcription may account for some of the interindividual variation in cytokine production. We have shown that a stable estimate of cytokine production phenotype can be obtained when monocytes collected on at least two separate occasions are stimulated by GM-CSF in vitro. We have looked for a relationship between IL-1 production and an 86-bp variable repeat polymorphism in intron 2 of the IL-1Ra gene. A less common allele of this polymorphism (allele 2) was associated with increased production of IL-1Ra protein, and also reduced production of IL-1 alpha protein by monocytes.

Cells, Cultured↗
PubMed1994

A rapid screening method for the detection of mutations in the RET proto-oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families.

Multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid carcinoma (FMTC) are autosomal dominant inherited cancer syndromes with incomplete penetrance. Following the identification of mutations in the RET proto-oncogene that segregate with the disease phenotype in MEN2A, MEN2B, and FMTC, genetic screening of individuals with mutations in RET may be performed. We have employed restriction endonuclease digestion of polymerase chain reaction products as an alternative to sequence analysis for rapid identification of mutant gene carriers in families in which MEN2A and FMTC are segregating. Twenty-one Australasian MEN2A and FMTC families have been screened for mutations in a cysteine-rich region of the RET proto-oncogene. Seven independent mutations were identified in key individuals in 16 of these families. We have identified a mutation in codon 620, 2053 T-->C (Cys620Arg), and two mutations in codon 634 of exon 11 of RET, 2095 T-->C (Cys634Arg) and 2096 G-->A (Cys634Tyr), all three of which were present in both MEN2A and FMTC families.

Australia↗
PubMed1994

Diverse phenotypes associated with exon 10 mutations of the RET proto-oncogene.

Mutations of the RET proto-oncogene are the underlying cause of some cases of Hirschsprung disease (HSCR) and the inherited cancer syndromes multiple endocrine neoplasia types 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). In HSCR these mutations are dispersed throughout the gene, while in MEN 2A and FMTC, they are tightly clustered in five cysteine codons of the RET extracellular domain. HSCR and MEN 2 are usually distinct but occasional families have been reported with both diseases. In each of five families with HSCR with or without MEN 2A or FMTC, we have identified a nucleotide substitution in one of the five cysteine codons previously associated with MEN 2A or FMTC. In one family, which had HSCR as its only phenotype, we detected a Cys-->Trp mutation at codon 609 which had not been previously observed. In three families, both HSCR and MEN 2A were associated with a single Cys-->Arg mutation at either codon 618 or 620 of RET. In the fifth family, FMTC and HSCR were present but we could not determine whether HSCR arose from mutation of the RET locus. We suggest that specific mutations in cysteine codons 618 and 620 result in MEN 2A or FMTC, but can also predispose to HSCR with low penetrance.

Base Sequence↗
PubMed1994

Effect of gold sodium thiomalate and its thiomalate component on the in vitro expression of endothelial cell adhesion molecules.

Endothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.

Animals↗
PubMed1993

Secretory leucocyte proteinase inhibitor is produced by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes.

The objective of this study was to examine the expression of the secretory leucocyte proteinase inhibitor (SLPI) gene by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes. RNA was extracted from human articular cartilage chondrocytes, synovial fibroblasts and fibrochondrocytes of the annulus fibrosus. Expression was analysed by Northern blotting and hybridisation to a SLPI RNA probe. SLPI was isolated from chondrocyte culture supernatant by gel-permeation and cation-exchange chromatography and examined by a solid-phase enzyme-linked immunofiltration assay for SLPI and by SDS/PAGE using Western blotting with polyclonal IgG samples against bronchial mucus leucocyte proteinase inhibitor and SLPI. This identified biochemically and immunologically the major serine proteinase inhibitory protein synthesised by chondrocytes as SLPI. Secretory leucocyte proteinase inhibitor mRNA was detected in RNA preparations from articular cartilage chondrocytes and annulus fibrosus fibrochondrocytes. The results presented here allowed us to deduce that human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes synthesized SLPI whereas human synovial fibroblasts did not.

Adult↗