A complex endocrine conundrum.

We describe a case of recurrent primary hyperparathyroidism, manifested as 3 metachronous parathyroid adenomata, in a 50 year-old woman who also had Hashimoto hypothyroidism, gastric gastrointestinal stromal tumour (GIST), cysts in liver and kidneys, 5 intestinal polyps (one of these a villous adenoma), diverticulitis and telangiectasia of lips. She did not have medullary thyroid carcinoma (MTC). Genetic analysis of the CDC73 gene [for Hyperparathyroidism-jaw tumor (HPT-JT)], MEN1 for Multiple Endocrine Neoplasia Type1, CDKN1B for MEN4, SDHB and SDHD for Paraganglioma/Pheochromocytoma susceptibility, VHL for von Hippel-Lindau Syndrome, BMPR1A and SMAD4 for Juvenile Polyposis Syndrome (JPS) (sequencing and MLPA), karyotype and array CGH (44 K) were all normal. She was found to be homozygous for a synonomous germline variant in exon 14 (p. Ser836Ser) of the RET oncogene. This RET variant is of unclear clinical significance, and has been previously reported both in normal individuals and in individuals with MTC. It is unlikely that homozygosity for the RET variant has been casual in the multiple pathologies that our patient has developed.

Parafibromin mutations in hereditary hyperparathyroidism syndromes and parathyroid tumours.

OBJECTIVE: To investigate two patients with the hyperparathyroidism-jaw tumour (HPT-JT) syndrome and three patients with familial isolated hyperparathyroidism (FIHP), together with 31 parathyroid tumours (2 HPT-JT, 2 FIHP and 27 sporadic) for HRPT2 mutations. The HPT-JT syndrome and FIHP are autosomal dominant disorders that may be caused by abnormalities of the HRPT2 gene, located on chromosome 1q31.2. HRPT2 encodes a 531 amino acid protein, parafibromin, which interacts with human homologues of the yeast Paf1 complex. DESIGN: Leukocyte and tumor DNA was used with HRPT2-specific primers for polymerase chain reaction amplification of the 17 exons and their splice junctions, and the DNA sequences of the polymerase chain reaction products determined. RESULTS: Three heterozygous germline HRPT2 mutations, two in HPT-JT and one in FIHP patients, were identified. These consisted of one 1-bp duplication (745dup1bp), 1 nonsense (Arg234Stop) and 1 missense (Asp379Asn) mutation. One parathyroid tumour from an FIHP patient was demonstrated to harbour a germline deletion of 1 bp together with a somatic missense (Leu95Pro) mutation, consistent with a 'two-hit' model for hereditary cancer. The 27 sporadic benign parathyroid tumours did not harbour any HRPT2 somatic mutations. Six HRPT2 polymorphisms with allele frequencies ranging from 2% to 15% were detected. CONCLUSIONS: Our results have identified three novel HRPT2 mutations (two germline and one somatic). The Asp379Asn mutation is likely to disrupt interaction with the human homologue of the yeast Paf1 complex, and the demonstration of combined germline and somatic HRPT2 mutations in a parathyroid tumour provide further evidence for the tumour suppressor role of the HRPT2 gene.

Refining the Amsterdam Criteria and Bethesda Guidelines: testing algorithms for the prediction of mismatch repair mutation status in the familial cancer clinic.

PURPOSE: Hereditary nonpolyposis colon cancer (HNPCC) is a Mendelian dominant syndrome of bowel, endometrial, and other cancers and results from germline mutations in mismatch repair (MMR) genes. HNPCC is now best diagnosed on molecular grounds using MMR mutation screening, aided by microsatellite instability (MSI) and immunohistochemistry in tumors. Selection of families for molecular investigation of HNPCC is usually based on suboptimal methods (Amsterdam Criteria or Bethesda Guidelines), but these can be improved using additional clinical data (mean ages of affected persons and presence of endometrial cancer) in a quantitative model. METHODS: We have verified the performance of the Wijnen model and have shown that it remains valid when HNPCC is diagnosed using mutation screening, MSI, and immunohistochemistry. We have also set up and verified our own models (Amsterdam-plus and Alternative), which perform at least as well as the Wijnen model. RESULTS: The Amsterdam-plus model improves on the Amsterdam Criteria by using five extra variables (numbers of colorectal and endometrial cancers in the family, number of patients with five or more adenomas, number with more than one primary cancer of the colorectum or endometrium, and mean age of presentation) and performs better than the Wijnen model. The Alternative model avoids the need to evaluate the Amsterdam Criteria and performs nearly as well as the other models. CONCLUSION: We believe that a quantitative model, such as the Amsterdam-plus model, should be the first choice for selecting families or patients for evaluation of HNPCC using molecular tests. We present an algorithm for this process.

How reliable is the allopurinol load in detecting carriers for ornithine transcarbamylase deficiency?

The allopurinol test aims to distinguish carriers and noncarriers for ornithine transcarbamylase (OTC) deficiency. We have evaluated the reliability of the test in at-risk females of known genotype. Results based on urine orotidine and/or orotic acid measurement were compared in terms of sensitivity and specificity. Retrospectively, we analysed the results of allopurinol tests in 42 women (22 confirmed heterozygotes and 20 noncarriers) from 23 pedigrees at risk of being carriers for OTC deficiency. Using a cut-off of 2 standard deviations above the mean of controls, the highest sensitivity (91%) was given by orotidine alone or in combination with orotic acid, but specificity was only 70% and 65%, respectively. We conclude that the value of the allopurinol test for detecting OTC carriers in at-risk females is limited. This needs to be recognized when counselling families. The test still has a role as a safe, quick, noninvasive screen of individuals at risk, but test results in possible carriers should be interpreted with caution. In the absence of other supportive evidence, confirmation by mutation analysis is required.

Outcomes following physical restraint reduction programs in two acute care hospitals.

BACKGROUND: Physical restraint rates can be reduced safely in long term care settings, but the strategies used to prevent wandering, falls, and patient aggression have not been tested for their effectiveness in preventing therapy disruption. A restraint reduction program (RRP) consisting of four core components (administrative, educational, consultative, and feedback) was implemented in 1998-1999 in 14 units at two acute care hospitals in geographically distant cities. METHODS: The RRP was targeted at units with prevalence rates of > or = 4% for non-intensive care units (non-ICUs) and > or = 25% for ICUs, as well as two additional units. The RRP was implemented by an interdisciplinary team consisting of geriatricians and nurse specialists. RESULTS: Of the 16,605 admissions to the RRP units, 2,772 cases received RRP consultations. Only six units (four of seven general units and two of six ICUs) demonstrated a relative reduction of > or = 20% in the physical restraint use rate. No increase in secondary outcomes of patient falls and therapy disruptions (patient-initiated discontinuation or dislodgment of therapeutic devices) occurred, injury rates were low, and no deaths occurred as a direct result of either a fall or therapy disruption event. DISCUSSION: Given the minimal success in the ICU settings, further studies are needed to determine effective nonrestraint strategies for critical care patients. ICU clinicians need to be persuaded of the favorable risk-to-benefit ratio of alternatives to physical restraint before they will change their practice patterns. SUMMARY: Efforts to identify more effective interventions that match patient needs and to identify non-clinician factors that affect physical restraint use are needed.

A common 2 bp deletion mutation in the glucose-6-phosphatase gene in Indian patients with glycogen storage disease type Ia.

This study reports a novel mutation which may be prevalent in Indian patients with glycogen storage disease type Ia.

Mutations in severe combined immune deficiency (SCID) due to JAK3 deficiency.

During the last 10 years, an increasing number of genes have been identified whose abnormalities account for primary immunodeficiencies, with defects in development and/or function of the immune system. Among them is the JAK3-gene, encoding for a tyrosine kinase that is functionally coupled to cytokine receptors which share the common gamma chain. Defects of this gene cause an autosomal recessive form of severe combined immunodeficiency with almost absent T-cells and functionally defective B-cells (T(-)B(+) SCID). Herewith, we present molecular information on the first 27 unique mutations identified in the JAK3 gene, including clinical data on all of the 23 affected patients reported so far. A variety of mutations scattered throughout all seven functional domains of the protein, and with different functional effects, have been identified. Availability of a molecular screening test, based on amplification of genomic DNA, facilitates the diagnostic approach, and has permitted recognition that JAK3 deficiency may also be associated with atypical clinical and immunological features. Development of a structural model of the JAK3 kinase domain has allowed characterization of the functional effects of the various mutations. Most importantly, molecular analysis at the JAK3 locus results in improved genetic counseling, allows early prenatal diagnosis, and prompts appropriate treatment (currently based on hematopoietic stem cell transplantation) in affected families.

Eleven novel JAK3 mutations in patients with severe combined immunodeficiency-including the first patients with mutations in the kinase domain.

Defects of the JAK3-gene are known to cause an autosomal recessive form of severe combined immunodeficiency with almost absent T-cells and functionally defective B-cells (T-B+SCID). The JAK3 protein, an intracellular tyrosine kinase, is crucial for signal-transmission from the common gamma chain to the Signal Transducers and Activators of Transcription (STATs) that drive gene expression in the nucleus. We present nine novel patients with eleven distinct mutations (g.96A>G, g.268G>C, IVS12-1G>A, g.2046C>T, g.2160C>T, g.2175G>A, g.2187G>T, g.2391C>T, g.2406C>T, IVS18+3G>C) among them a mutation in the kinase domain (JH1: g.3167del). The clinical phenotype of the patients shows an unusually broad spectrum ranging from classical SCID to almost normal. In order to understand the complex genotype-phenotype correlation we studied expression and function (by IL-2 induced phosphorylation) of the newly identified and two other alleles with JH1 mutations we recently reported. We found the first mutation in the JH1-domain of JAK3, that precludes kinase activity (L910S). The two other JH1 mutations both caused a premature stop. One of them (C1024fsX1037) also abolished any phosphorylation of JAK3 and expression of the protein. The other mutation (Y1023X), affecting the last JH1 tyrosine, may allow for residual protein expression and phosphorylation. This may indicate that the part of the kinase region downstream Y1023, is not essential for the function of JAK3.

X-linked lymphoproliferative disease: three atypical cases.

Common variable immunodeficiency (CVID) is the most frequently occurring primary immunodeficiency in both children and adults. The molecular basis of CVID has not been defined, and diagnosis involves exclusion of other molecularly defined disorders. X-linked lymphoproliferative disease (XLP) is a rare disorder in which severe immunodysregulatory phenomena typically follow Epstein-Barr virus (EBV) infection. Boys who survive initial EBV infection have a high incidence of severe complications, including progressive immunodeficiency, aplastic anaemia, lymphoproliferative disease and lymphoma. Survival beyond the second decade is unusual, although bone marrow transplantation can be curative. Until recently reliable diagnostic testing for XLP has not been available, but the identification of the XLP gene, known as SH2D1A, and coding for a protein known as SAP, means that molecular diagnosis is now possible, both by protein expression assays, and mutation detection, although the mutation detection rate in several series is only 55-60%. We describe three male patients initially diagnosed as affected by CVID, one of whom developed fatal complications suggestive of XLP, and all of whom lack expression of SAP. Two out of three have disease-causing mutations in the SAP gene, consistent with published data for XLP. These findings raise the possibility that a subgroup of patients with CVID may be phenotypic variants of XLP. Further studies are necessary to investigate this possibility, and also to clarify the prognostic significance of SAP abnormalities in such patients in the absence of typical features of XLP.

Defective expression of the interleukin-2/interleukin-15 receptor beta subunit leads to a natural killer cell-deficient form of severe combined immunodeficiency.

Development of T and natural killer (NK) cells is critically dependent on cytokine signaling, and defects in cytokine receptor complex subunits have been shown to result in severe combined immunodeficiency (SCID) syndromes in humans and in murine models. An infant boy had typical clinical features of SCID and was found to lack NK cells in his peripheral circulation. Molecular analysis did not reveal abnormalities in his gammac or JAK-3 genes, and he was investigated for defects in the interleukin-15 (IL-15) receptor complex because functional IL-15 signaling is essential for NK cell development. Expression of the IL-2R/IL-15Rbeta chain was significantly reduced in the patient's peripheral blood mononuclear cells (PBMCs) by immunoblot, flow cytometry, and Northern blot analysis. Furthermore, IL-2 stimulation of PBMCs showed only minimal tyrosine phosphorylation of JAK-3. These data demonstrate that defects in IL-2R/1L-15Rbeta expression can lead to a unique NK-deficient SCID immunophenotype. (Blood. 2001;98:877-879)

Protein assays for diagnosis of Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

Mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) give rise to Wiskott-Aldrich syndrome (WAS), a condition that exhibits a wide spectrum of clinical severity. Patients may develop mild thrombocytopenia or suffer from a wide range of associated disorders including eczema, immune dysfunction, autoimmune disease and malignancy. The clinical diagnosis of Wiskott-Aldrich syndrome (WAS) can be difficult and is usually supported by the detection of WASp gene mutations using genetic analysis. Recently, protein-based assays have been used to demonstrate the absence of WASp in patients known to have WASp gene mutations. We have now reversed this approach and report on the use of immunoblot assays to rapidly diagnose WAS in 13 patients. There was a complete absence of WASp in 10 out of 13 patients and an abnormal protein form was detected in the remaining three patients. In all cases, subsequent genetic analysis confirmed the presence of a WASp gene mutation. We believe that protein-based assays should be employed as the first line of investigation in the diagnosis of WAS spectrum disorders.

Rapid protein-based assays for the diagnosis of T-B+ severe combined immunodeficiency.

The severe combined immunodeficiencies (SCID) are a heterogeneous group of conditions arising from a variety of molecular defects. The X-linked form of SCID (X-SCID) is caused by defects in the common gamma chain (gammac), and is characterized by a T-B+NK- immunophenotype. This lymphocyte profile is seen in an autosomal recessive form of SCID caused by mutations in the JAK3 molecule. Thus, X-SCID and JAK3-deficient SCID are clinically and immunologically indistinguishable. Knowledge of the precise molecular defect is essential for antenatal diagnosis, carrier testing and for treatment using somatic gene therapy. To identify the molecular defect in children presenting with a T-B+NK- form of SCID, we have developed rapid assays based on flow cytometric analysis of gammac, immunoblotting for JAK3 and gammac, and detection of interleukin-2 (IL-2)-induced tyrosine phosphorylation of JAK3. Sixteen T-B+NK- SCID patients from 15 families were examined. Nine had no detectable gammac, four had abnormal gammac expression and no IL-2-induced JAK3 tyrosine phosphorylation, and one had normal gammac expression but no IL-2-induced JAK3 tyrosine phosphorylation, although JAK3 was present. All these patients had mutations identified in their gammac gene. Two patients exhibited normal gammac expression, but JAK3 was not detected by immunoblotting and these patients were confirmed as having JAK3 gene mutations. Thus, these protein-based assays have led to rapid molecular diagnoses in T-B+ SCID that have subsequently been confirmed by genetic analysis.

Mutation detection in 65 families with a possible diagnosis of ornithine carbamoyltransferase deficiency including 14 novel mutations.

The high new mutation rate and the wide spectrum of mutations found in patients with ornithine carbamoyltransferase (OCT) deficiency means that direct mutation analysis is essential for providing accurate carrier detection and prenatal diagnosis in affected families. We present our strategy for mutation detection in the OCT gene and summarize the results from 31 families with a confirmed diagnosis and 34 families with a suspected diagnosis of OCT deficiency, and describe 14 previously unreported mutations.

Diagnosis of X-linked lymphoproliferative disease by analysis of SLAM-associated protein expression.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency in which affected boys show abnormal responses to Epstein-Barr virus infection. The gene defective in XLP has been identified and designated SH2D1A and encodes a protein termed SLAM-associated protein (SAP). Mutation analysis in individuals with typical XLP presentations and family histories has only detected abnormalities in approximately 60% of patients. Thus, genetic analysis alone cannot confirm a diagnosis of XLP We have developed a SAP expression assay that can be used as a diagnostic indicator of XLP We show that SAP is constitutively expressed in normal individuals, in patients with severe sepsis and in patients with other primary immunodeficiencies. In six XLP patients, four with classical and two with atypical presentations, SAP expression was absent. In the latter two, who were previously assigned as having common variable immunodeficiency (CVID), the diagnosis of XLP was initially made using the protein expression assay. In two further patients in whom no mutation could be detected by genetic analysis, lack of SAP expression strongly suggests that these individuals have XLP. We therefore suggest that XLP should be suspected in certain boys previously diagnosed as having CVID and recommend that patients are investigated both by genetic analysis of SH2D1A and by expression of SAP protein.

The effect of exercise training on the severity and duration of a viral upper respiratory illness.

PURPOSE: The purpose of this investigation was to determine whether exercise training affects the severity and duration of a rhinovirus-caused upper respiratory illness (URI). METHODS: Subjects who were rhinovirus 16 (RV 16) antibody-free completed a graded exercise test. Thirty-four individuals (ages 18-29 yr) of moderate fitness (32 mL.kg-1.min-1 to 60 mL.kg-1.min-1) were randomly assigned to the exercise group (EX) while 16 additional individuals of similar age and fitness served as a nonexercise (NEX) control group. All EX and NEX subjects were inoculated with RV 16 on 2 consecutive days. EX subjects completed 40 min of supervised exercise every other day at 70% of heart rate (HR) reserve for a 10-d period. Every 12 h, all subjects completed a 13-item symptom severity checklist and a physical activity log. Used facial tissues were collected and weighed (symptom severity measure) during these same reporting periods. RESULTS: A two group by nine measure (2 x 9) repeated measures ANOVA procedure showed no difference in symptom questionnaire mean scores and the mucous weights of the EX and NEX groups for days 2-10 of the experiment. A two measure by five measure (2 x 5) repeated measures ANOVA procedure indicated no differences between the pre- and post-exercise questionnaire means for the five sessions that EX subjects exercised. Statistical significance was set at P < 0.05. CONCLUSION: These results suggest that moderate exercise training during a rhinovirus-caused URI under the conditions of this study design do not alter the severity and duration of the illness.

Soluble interleukin-2 receptor is a thyroid hormone-dependent early-response marker in the treatment of thyrotoxicosis.

Thyrotoxic patients exhibit increased levels of immune activation molecules (soluble interleukin-2 receptor [sIL-2R], intercellular adhesion molecule-1 [ICAM-1], and endothelial-leukocyte adhesion molecule-1 [ELAM-1]) in serum, although the clinical significance of these measurements remains unclear. In a randomized 4-week study, we have recently shown that in the treatment of hyperthyroidism, the combination of cholestyramine and methimazole (MMI) resulted in faster lowering of serum thyroid-hormone levels than did MMI alone. Stored serial serum samples from patients participating in this randomized treatment trial were analyzed for sIL-2R, soluble ICAM-1 (sICAM-1), and soluble ELAM-1 (sELAM-1). The levels of all three molecules were elevated in patients with hyperthyroidism. Although the levels of sICAM-1 and sELAM-1 remained elevated through the 4-week follow-up period in both groups of patients, the sIL-2R levels (normal levels, 1.0 to 4.2 ng/ml) decreased significantly in the 10 patients who received cholestyramine in addition to MMI (week 0, 14.2 +/- 1.5 ng/ml; week 2, 10.8 +/- 1.2 ng/ml; week 4, 8.9 +/- 1.5 ng/ml). In eight patients who received MMI alone, sIL-2R decreased less rapidly (week 0, 12.3 +/- 1.4 ng/ml; week 2, 12.3 +/- 1.3 ng/ml; week 4, 10.9 +/- 1.3 ng/ml). sICAM-1 and sELAM-1 were elevated at baseline but did not decrease during therapy. In the former group, free thyroxine and free triiodothyronine decreased faster. These data show that levels of sIL-2R in serum, but not those of sICAM-1 and sELAM-1, may be of clinical use in the early follow-up evaluation of medically treated patients.