Janet Warner

Genetic testing in familial isolated hyperparathyroidism: unexpected results and their implications

Familial hyperparathyroidism is not uncommon in clinical endocrine practice. It encompasses a spectrum of disorders including multiple endocrine neoplasia types 1 (MEN1) and 2A, hyperparathyroidism-jaw tumour syndrome (HPT-JT), familial hypocalciuric hypercalcaemia (FHH), and familial isolated hyperparathyroidism (FIHP). Distinguishing among the five syndromes is often difficult but has profound implications for the management of patient and family. The availability of specific genetic testing for four of the syndromes has improved diagnostic accuracy and simplified family monitoring in many cases but its current cost and limited accessibility require rationalisation of its use. No gene has yet been associated exclusively with FIHP. FIHP phenotypes have been associated with mutant MEN1 and calcium-sensing receptor (CASR) genotypes and, very recently, with mutation in the newly identified HRPT2 gene. The relative proportions of these are not yet clear. We report results of MEN1, CASR, and HRPT2 genotyping of 22 unrelated subjects with FIHP phenotypes. We found 5 (23%) with MEN1 mutations, four (18%) with CASR mutations, and none with an HRPT2 mutation. All those with mutations had multiglandular hyperparathyroidism. Of the subjects with CASR mutations, none were of the typical FHH phenotype. These findings strongly favour a recommendation for MEN1 and CASR genotyping of patients with multiglandular FIHP, irrespective of urinary calcium excretion. However, it appears that HRPT2 genotyping should be reserved for cases in which other features of the HPT-JT phenotype have occurred in the kindred. Also apparent is the need for further investigation to identify additional genes associated with FIHP.

A report of a national mutation testing service for the MEN1 gene: clinical presentations and implications for mutation testing

Introduction: Mutation testing for the MEN1 gene is a useful method to diagnose and predict individuals who either have or will develop multiple endocrine neoplasia type 1 (MEN 1). Clinical selection criteria to identify patients who should be tested are needed, as mutation analysis is costly and time consuming. This study is a report of an Australian national mutation testing service for the MEN1 gene from referred patients with classical MEN 1 and various MEN 1-like conditions. Results: All 55 MEN1 mutation positive patients had a family history of hyperparathyroidism, had hyperparathyroidism with one other MEN1 related tumour, or had hyperparathyroidism with multiglandular hyperplasia at a young age. We found 42 separate mutations and six recurring mutations from unrelated families, and evidence for a founder effect in five families with the same mutation. Discussion: Our results indicate that mutations in genes other than MEN1 may cause familial isolated hyperparathyroidism and familial isolated pituitary tumours. Conclusions: We therefore suggest that routine germline MEN1 mutation testing of all cases of “classical” MEN1, familial hyperparathyroidism, and sporadic hyperparathyroidism with one other MEN1 related condition is justified by national testing services. We do not recommend routine sequencing of the promoter region between nucleotides 1234 and 1758 (Genbank accession no. U93237) as we could not detect any sequence variations within this region in any familial or sporadic cases of MEN1 related conditions lacking a MEN1 mutation. We also suggest that testing be considered for patients <30 years old with sporadic hyperparathyroidism and multigland hyperplasia.

Familial isolated hyperparathyroidism is linked to a 1.7 Mb region on chromosome 2p13.3–14

Bachground: Familial isolated hyperparathyroidism (FIHP) is an autosomal dominantly inherited form of primary hyperparathyroidism. Although comprising only about 1% of cases of primary hyperparathyroidism, identification and functional analysis of a causative gene for FIHP is likely to advance our understanding of parathyroid physiology and pathophysiology. Methods: A genome-wide screen of DNA from seven pedigrees with FIHP was undertaken in order to identify a region of genetic linkage with the disorder. Results: Multipoint linkage analysis identified a region of suggestive linkage (LOD score 2.68) on chromosome 2. Fine mapping with the addition of three other families revealed significant linkage adjacent to D2S2368 (maximum multipoint LOD score 3.43). Recombination events defined a 1.7 Mb region of linkage between D2S2368 and D2S358 in nine pedigrees. Sequencing of the two most likely candidate genes in this region, however, did not identify a gene for FIHP. Conclusions: We conclude that a causative gene for FIHP lies within this interval on chromosome 2. This is a major step towards eventual precise identification of a gene for FIHP, likely to be a key component in the genetic regulation of calcium homeostasis.

High incidence of macrotroponin I with a high-sensitivity troponin I assay

Abstract Background: Cardiac troponin is the preferred biomarker of myocardial injury. High-sensitivity troponin assays allow measurement of very low levels of troponin with excellent precision. After the introduction of a high-sensitivity troponin I assay the laboratory began to receive enquiries from clinicians about clinically discordant elevated troponin I results. This led to a systematic investigation and characterisation of the cause. Methods: Routine clinical samples were measured by the Architect High Sensitive Troponin-I (hsTnI) and the VITROS Troponin I ES assays (VitrosTnI). Results that were elevated according to the Architect but not the VITROS assay (Group 1) or results elevated by both assays but disproportionately higher on the Architect (Group 2) were re-analysed for hsTnI after re-centrifugation, multiple dilutions, incubation with heterophilic blocking reagents, polyethylene glycol (PEG) precipitation, and Protein A/G/L treatment. Sephacryl S-300 HR gel filtration chromatography (GFC) was performed on selected specimens. Results: A high molecular weight complex containing immunoreactive troponin I and immunoglobulin (macrotroponin I) was identified in 5% of patients with elevated hsTnI. Patients with both macrotroponin and myocardial injury had higher and longer elevation of hsTnI compared with VitrosTnI with peaks of both macrotroponin and free troponin I-C complex on GFC. Conclusions: Circulating macrotroponin I (macroTnI) causes elevated hsTnI results with the Architect High Sensitive Troponin-I assay with the potential to be clinically misleading. The assay involved in this investigation may not be the only assay affected by macrotroponin. It is important for laboratories and clinicians to be aware of and develop processes to identify and manage specimens with elevated results due to macrotroponin.

17-hydroxyprogesterone quantification in dried blood spots by high performance liquid chromatography-tandem mass spectroscopy

Measurement of 17-hydroxyprogesterone (17-OHP) in daily dried blood spot profiles via radioimmunoassay is a convenient and accepted method for monitoring of glucocorticoid therapy in Congenital Adrenal Hyperplasia. Characteristics of this method that serve to limit its clinical usefulness include its lack of specificity for 17-OHP, and a relatively high limit of detection. Mass Spectroscopy is the gold standard method for steroid quantification. We aim to establish a High Performance Liquid Chromatography-Tandem Mass Spectroscopy (HPLC-MS/MS) method for dried blood spot 17-OHP quantification and develop normative age- and tanner-specific, as well as Congenital Adrenal Hyperplasia genotype-phenotype correlated reference ranges to guide glucocorticoid therapy. Four 3mm blood spots were punched from patient dried blood spot filter paper specimens. 17-OHP was eluted into solvent and concentrated using liquid nitrogen. Steroids were separated using high performance liquid chromatography and quantitated by Tandem Mass Spectrometry. For the radioimmunassay / HPLC-MS/MS correlations, measurements were performed by both methods on 49 samples from children with glucocorticoid-dependent Congenital Adrenal Hyperplasia, as well as children undergoing dynamic endocrine function testing at The Mater Children’s Hospital. Reference samples for HPLC-MS/MS calibration, and determination of sensitivity, precision, and recovery were prepared using whole blood samples spiked with 17-OHP spotted onto filter paper. Concentrations were expressed as mean (nmol/L), standard deviation, and coefficient of variation (%). There was excellent correlation between HPLC-MS/MS and radioimmunoassay methods (r2=0.9610). For the radioimmunoassay method, the lower limit of quantification has been established at <5 nmol/L while LC-MS/MS allows detection to 1.0 nmol/L (1.2nmol/L, 0.26, 22%) with a proposed lower limit of quantification of 1.5 nmol/L (1.3 nmol/L, 1.3, 13%). We have established an accurate, reliable, and specific method for quantifying 17-OHP concentrations from dried blood spot using HPLC-MS/MS. This will allow for the establishment of clinically relevant and time-specific reference ranges to guide glucocorticoid dose adjustment in children with Congenital Adrenal Hyperplasia.

Internet slimming, thyrotoxicosis and the liver.

The Medical Journal of Australia ISSN: 0025729X 21 April 2014 200 7 419-420 ©The Medical Journal of Australia 2014 www.mja.com.au Lessons from Practice significant pharmacological efficacy as loss, it is not surprising that adulterati tion slimming products with pharmaco stances occurs. Desiccated porcine a tissue, a past treatment for hypothyroid through compounding pharmacies a purchased online. Thyrotoxicosis cau animal thyroid tissue in minced beef ments has been reported.2,3 In the pre erb ov cac Administra H al dietary supplements are readily available er the internet with no regulation of their effiy, safety or contents. The Therapeutic Goods tion has issued a warning against the importation and use of these products.1 However, with convenience, anonymity, lower cost and, perhaps, ignorance of the risks, the practice persists and its scale is unknown. Given that no herbal ingredient has been shown to have an agent of weight on of non-prescriplogically active subnd bovine thyroid ism, is still available nd can be readily sed by ingestion of and herbal supplesent case, the addition of desiccated raw thyroid to the plant material in the capsules was probably the cause of thyrotoxicosis. Although no reports of infectious disease following the consumption of dried raw animal tissue from herbal supplements could be found in the literature, contracting an infection is possible — for example, due to spore-forming pathogens. A recent Australian report on the presence of thyroid tissue in a dietary supplement3 also measured a significant amount of thyroid-stimulating hormone in the material, suggesting the additional presence of pituitary tissue. Although the risk of developing variant Creutzfeldt–Jakob disease from ingestion of bovine spongiform encephalopathy-infected tissue is probably low in this setting, it is possible.4 The adulteration of dietary supplements and herbal medicines with synthetic drugs was first described over 10 years ago. Adulterants have included steroids, opiates, with palpitations, tremor and fatigue. Investigations revealed mild thyrotoxicosis, with a free thyroxine level of 30 pmol/L (reference interval [RI], 10–20 pmol/L), a free triiodothyronine level of 16.9 pmol/L (RI, 2.8–6.8 pmol/L) and a thyroidstimulating hormone level < 0.05 mIU/L (RI, 0.4–4 mIU/L). Liver enzyme levels were mildly abnormal — alkaline phosphatase, 121 U/L (RI, 30–115 U/L); -glutamyl transferase, 134 U/L (RI, 0–45 U/L); and alanine aminotransferase, 49 U/L (RI, 0–45 U/L) — except for aspartate aminotransferase, 26 U/L (RI, 0–41 U/L). The patient denied neck pain and intake of medications, alcohol or iodine. Her medical and family histories were unremarkable, and body mass index was 29.4 kg/m2. The thyroid was normal to palpation with no bruit. There was no ophthalmopathy or dermopathy. Gastrointestinal examination was unremarkable. Further investigations included negative test results for thyroid-stimulating hormone receptor, antithyroglobulin and antithyroid peroxidase antibodies, normal thyroid ultrasound and colour-flow Doppler results, and low– normal uptake throughout both thyroid lobes on technetium99m nuclear medicine scan. Viral and autoimmune hepatitis, haemochromatosis, Wilson disease and -1 antitrypsin deficiency were excluded as the cause of hepatitis. Liver ultrasound was unremarkable.

Symptomatic hypocalcemia secondary to rifampicin-induced hypovitaminosis D

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Recovery of Drugs of Abuse from Dräger DCD5000 Oral Fluid Collection Device in Australia

In Australia, it is a requirement of workplace oral fluid (OF) drugs of abuse testing that drug recovery from collection devices be verified by an accredited laboratory. Recovery data are used in conjunction with collection volume imprecision data and uncertainty of measurement to provide an estimation of drug concentration in neat OF. The manufacturers product information for the DCD5000 collection device indicates that the collection volume of the swab is 380 µL. Recovery data for the swab when used with the isopropanol provided by the manufacturer are not available. A series of experiments using fortified drug-free OF were performed to assess the collection volume imprecision of the Dräger DCD5000 swab and the recovery of drugs from the swab using isopropanol. The fortified OF was collected with the swabs (n = 16), and swabs were discharged into vials of isopropanol as per the manufacturers instructions. The mean collection volume of the DCD5000 swab was 487 µL with an imprecision of 1.3%. Recovery of drug from the device ranged from 86 to 98% for drugs listed in the Australian OF workplace testing standard. Recovery of methadone, buprenorphine and norbuprenorphine ranged from 93 to 102%. Recovery of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene was 45%, suggesting that urine is more suitable sample if methadone therapy is being monitored. Overall, drug recovery from the device using isopropanol was acceptable when the increased collection volume of the swab was taken into account.