Lucia Gagliardi

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Corticosteroid-binding globulin: the clinical significance of altered levels and heritable mutations.

Corticosteroid-binding globulin (CBG) is the specific high-affinity plasma transport glycoprotein for cortisol. Stress-induced falls in CBG levels may heighten hypothalamic-pituitary-adrenal axis responses and CBG:tissue interactions may allow targeted cortisol delivery. Three genetic variants of CBG have been identified that reduce cortisol binding affinity and/or CBG levels. These include the Leuven and Lyon mutations which reduce CBG:cortisol binding affinity 3- and 4-fold, respectively, and the null mutation resulting in a 50% (heterozygote) or 100% (homozygote) reduction in CBG levels. The three reported null homozygotes demonstrate that complete CBG deficiency is not lethal, although it may be associated with hypotension and fatigue. The phenotype of a CBG null murine model included fatigue and immune defects. One community-based study revealed that severe CBG mutations are rare in idiopathic fatigue disorders. The mechanisms by which CBG mutations may cause fatigue are unknown. There are preliminary data of altered CBG levels in hypertension and in the metabolic syndrome; however, the nature of these associations is uncertain. Further studies may clarify the functions of CBG, and clinical observations may validate and/or extend the phenotypic features of various CBG mutations.

ARMC5 Mutations Are Common in Familial Bilateral Macronodular Adrenal Hyperplasia

CONTEXT Bilateral macronodular adrenal hyperplasia (BMAH) is a rare form of adrenal Cushings syndrome. Familial cases have been reported, but at the time we conducted this study, the genetic basis of BMAH was unknown. Recently, germline variants of armadillo repeat containing 5 (ARMC5) in patients with isolated BMAH and somatic, second-hit mutations in tumor nodules, were identified. OBJECTIVE Our objective was to identify the genetic basis of familial BMAH. DESIGN We performed whole exome capture and sequencing of 2 affected individuals from each of 4 BMAH families (BMAH-01, BMAH-02, BMAH-03, and BMAH-05). Based on clinical evaluation, there were 7, 3, 3, and 4 affected individuals in these families, respectively. Sanger sequencing of ARMC5 was performed in 1 other BMAH kindred, BMAH-06. RESULTS Exome sequencing identified novel variants Chr16:g.31477540, c.2139delT, p.(Thr715Leufs*1) (BMAH-02) and Chr16:g.31473811, c.943C→T, p.(Arg315Trp) (BMAH-03) in ARMC5 (GRch37/hg19), validated by Sanger sequencing. BMAH-01 had a recently reported mutation Chr16:g.31476121, c.1777C→T, p.(Arg593Trp). Sanger sequencing of ARMC5 in BMAH-06 identified a previously reported mutation, Chr16:g. 31473688; c.799C→T, p.(Arg267*). The genetic basis of BMAH in BMAH-05 was not identified. CONCLUSIONS Our studies have detected ARMC5 mutations in 4 of 5 BMAH families tested, confirming that these mutations are a frequent cause of BMAH. Two of the 4 families had novel mutations, indicating allelic heterogeneity. Preclinical evaluation did not predict mutation status. The ARMC5-negative family had unusual prominent hyperaldosteronism. Further studies are needed to determine the penetrance of BMAH in ARMC5 mutation-positive relatives of affected patients, the practical utility of genetic screening and genotype-phenotype correlations.

Continuous Subcutaneous Hydrocortisone Infusion Therapy in Addison's Disease: A Randomized, Placebo-Controlled Clinical Trial

CONTEXT Patients with Addisons disease (AD) report impaired subjective health status (SHS). Since cortisol exhibits a robust circadian cycle that entrains other biological clocks, impaired SHS may be due to the noncircadian cortisol profile achieved with conventional glucocorticoid replacement. Continuous subcutaneous hydrocortisone infusion (CSHI) reproduces a circadian cortisol profile, but its effects on SHS have not been objectively evaluated. OBJECTIVE The aim of this study was to determine the effect of CSHI on SHS in AD. SETTING AND DESIGN This was a multicentre, double-blind, placebo-controlled trial of CSHI vs oral glucocorticoid therapy. Participants received in random order 4 weeks of: CSHI and oral placebo, and subcutaneous placebo and oral hydrocortisone, separated by a 2-week washout period. SHS was assessed using the Short-Form 36 (SF-36), General Health Questionnaire (GHQ-28), Fatigue Scale (FS), Gastrointestinal Symptom Rating Scale (GSRS); and Addisons Quality of Life Questionnaire (AddiQoL). Participants were asked their (blinded) treatment preference. Twenty-four hour urine free cortisol (UFC) and diurnal salivary cortisol collections compared cortisol exposure during each treatment. RESULTS Ten participants completed the study. Baseline SHS scores (mean ± SE) were consistent with mild impairment: SF-36 physical component summary 48.4 (± 2.4), mental component summary 53.3 (± 3.0); GHQ-28 18.1 (± 3.3); GSRS 3.7 (± 1.6), and AddiQoL 94.7 (± 3.7). FS was similar to other AD cohorts 13.5 (± 1.0) (P = 0.82). UFC between treatments was not different (P = 0.87). The salivary cortisol at 0800 h was higher during CSHI (P = 0.03), but not at any other time points measured. There was no difference between the treatments in the SHS assessments. Five participants preferred CSHI, four oral hydrocortisone, and one was uncertain. CONCLUSIONS Biochemical measurements indicate similar cortisol exposure during each treatment period, although a more circadian pattern was evident during CSHI. CSHI does not improve SHS in AD with good baseline SHS. This casts some doubt on the potential benefit of circadian cortisol delivery on SHS in AD.

Familial vasopressin-sensitive ACTH-independent macronodular adrenal hyperplasia (VPs-AIMAH): clinical studies of three kindreds.

Objective  Cushings syndrome due to familial ACTH‐independent macronodular adrenal hyperplasia (AIMAH) has been reported in small kindreds. In vasopressin‐sensitive AIMAH (VPs‐AIMAH), VP stimulates an aberrant, ACTH‐independent increase in cortisol. The aims of this study were to (i) delineate the preclinical phenotype of VPs‐AIMAH in a three‐generation kindred (AIMAH‐01) and two smaller kindreds (AIMAH‐02 and AIMAH‐03) and (ii) investigate the aetiology of VP sensitivity in AIMAH‐01.

Low-dose hydrocortisone replacement improves wellbeing and pain tolerance in chronic pain patients with opioid-induced hypocortisolemic responses. A pilot randomized, placebo-controlled trial.

UNLABELLED Long-term opioid therapy has been associated with low cortisol levels due to central suppression of the hypothalamic-pituitary-adrenal axis. The implications of hypocortisolism on wellbeing have not been established. Our aim was to determine whether intervention with physiologic glucocorticoid replacement therapy improves wellbeing and analgesic responses in patients with chronic non-cancer pain on long-term opioid therapy with mild cortisol deficiency. We performed a pilot randomized, double-blind, placebo-controlled crossover study of oral hydrocortisone replacement therapy in 17 patients recruited from a Pain Clinic at a single tertiary center in Adelaide, Australia. Patients were receiving long-term opioid therapy (≥ 20 mg morphine equivalents per day for ≥ 4 weeks) for chronic non-cancer pain with mild hypocortisolism, as defined by a plasma cortisol response ≤ 350 nmol/L at 60 min following a cold pressor test. The crossover intervention included 28-day treatment with either 10mg/m(2)/day of oral hydrocortisone in three divided doses or placebo. Improvement in wellbeing was assessed using Version 2 of the Short Form-36 (SF-36v2), Brief Pain Inventory-Short Form, and Addisons disease quality of life questionnaires; improvement in analgesic response was assessed using cold pressor threshold and tolerance times. Following treatment with hydrocortisone, the bodily pain (P=0.042) and vitality (P=0.013) subscales of the SF-36v2 were significantly better than scores following treatment with placebo. There was also an improvement in pain interference on general activity (P=0.035), mood (P=0.03) and work (P=0.04) following hydrocortisone compared with placebo. This is the first randomized, double-blind placebo-controlled trial of glucocorticoid replacement in opioid users with chronic non-cancer pain and mild hypocortisolism. Our data suggest that physiologic hydrocortisone replacement produces improvements in vitality and pain experiences in this cohort compared with placebo. TRIAL REGISTRATION Therapeutic Goods Administration Clinical Trials Notification Scheme (Drugs), Trial Number 2012/0476.

Allan-Herndon-Dudley syndrome with unusual profound sensorineural hearing loss.

The Allan–Herndon–Dudley syndrome is caused by mutations in the thyroid hormone transporter, Monocarboxylate transporter 8 (MCT8). It is characterized by profound intellectual disability and abnormal thyroid function. We report on a patient with Allan–Herndon–Dudley syndrome (AHDS) with profound sensorineural hearing loss which is not usually a feature of AHDS and which may have been due to a coexisting nonsense mutation in Microphthalmia‐associated transcription factor (MITF).

ARMC5 is not implicated in familial hyperaldosteronism type II (FH-II)

Germline loss-of-function mutations in the armadillo-repeat-containing 5 (ARMC5) gene are an established cause of Cushings syndrome due to bilateral macronodular adrenal hyperplasia (BMAH), 1,2 and may play a role in primary aldosteronism. 3 As familial hyperaldosteronism type II (FH-II) has a presumed genetic basis, 4 we hypothesised that germline ARMC5 mutations underlie FH-II. We interrogated whole-exome sequencing data from four FH-II families. We did not identify any pathogenic ARMC5 variants which segregated with the phenotype of primary aldosteronism.

Autosomal dominant hypocalcaemia due to a novel CASR mutation: clinical and genetic implications

We report a patient from a family with autosomal dominant hypocalcaemia who also had prednisolone-treated Langerhans’ cell histiocytosis and familial genetic studies identifying a novel calcium-sensing receptor (CASR) mutation. We describe an approach to define pathogenicity of a genetic variation of uncertain significance and postulate that bisphosphonates may have paradoxically increased fracture risk in our patient. In 2009, a 49-year-old woman (III:1; Fig. 1a) was referred with recurrent fractures. Past history included: (i) hypocalcaemia (age 30) – manifesting with fatigue, arthralgia and tetany; despite continuous calcitriol and calcium carbonate, she had hospitalizations with tetany and (ii) prednisolone-treated (minimum 15 mg daily) pulmonary Langerhans’ cell histiocytosis (age 44). She was a current smoker (20 pack years). Hormone replacement therapy was prescribed for menopausal symptoms since age 46. Family history revealed dominantly inherited hypocalcaemia (10 individuals spanning three generations) (Fig. 1a). Individuals generally presented in their thirties with mildly symptomatic hypocalcaemia and received calcitriol and calcium. There was no family history of minimal trauma fracture. In mid-2007, the patient fractured two ribs coughing. Lumbar spine T-score was 0 4 (+0 3 2 years earlier), and femoral neck was 0 3. Risedronate 35 mg weekly was commenced in October 2007. However, nine further minimal trauma fractures occurred prior to review. Imaging showed no lytic or mass lesions at fracture sites. Mild paraesthesiae occurred intermittently. Corrected calcium was 1 87 mmol/l [reference range (RR): 2 1–2 5 mmol/ l], phosphate 1 65 mmol/l (RR: 0 8–1 45 mmol/l), 25-hydroxy vitamin D 34 nmol/l (RR: 60–160 nmol/l), parathyroid hormone (PTH) 1 1 pmol/l (RR: 0 8–5 5 pmol/l), beta C-terminal telopeptide <70 ng/l (desirable range: <400 ng/l) and urinary calcium 9 2 mmol/24 h (RR: 2 5–7 5 mmol/24 h). Bone density was stable. Imaging showed basal ganglia, but not renal, calcification. She fractured three ribs manoeuvring during a bone scan, which showed no other pathology. She did not consent to a tetracycline-labelled iliac crest biopsy to further assess bone turnover. Cholecalciferol was added and calcium carbonate doubled to 600 mg twice daily, improving symptoms and biochemistry. Her respiratory physicians agreed to taper prednisolone. Approval for teriparatide was granted by our institutional Pharmacy Committee and risedronate ceased. Severe bony pain occurred 1 week after commencing teriparatide 20 lg daily. This resolved and recurred after cessation and reintroduction of teriparatide, respectively, which was withdrawn indefinitely. She then fractured three phalanges of her foot during a low trauma fall. Hormone replacement therapy ceased, and warfarin commenced after a pulmonary embolism. She received 5 mg zoledronic acid. One week later, she fractured a rib coughing. In 2010, she had three lower respiratory tract infections. She declined hospitalization for the third and died at home. Autopsy was not performed. Sanger sequencing of CASR performed by the Genetics Laboratories, Churchill Hospital, Oxford, UK, using germline DNA from an affected sibling (proband – genetic studies: III:2; Fig. 1a), identified a heterozygous three base pair in-frame deletion in the coding region for transmembrane domain 6 (c.2443_2445delTTC; p.Phe815del; gene sequence accession NM_000388.1). This was not in the CASR database (http://www.casrdb.mcgill.ca/), a population screen we performed (616 individuals) or the Exome Aggregation Consortium Browser (http://exac.broadinstitute.org/). Segregation analysis of the deletion and other candidate gene screening were performed. This study was approved by the Royal Adelaide Hospital Human Research Ethics Committee (Protocol: 110623); all subjects gave written consent. Whole exome capture and sequencing (Australian Genome Research Facility, Victoria) of the proband’s germline DNA was performed using Illumina TruSeq Capture protocol and HiSeq 2000 platform (Illumina, Inc, San Diego, CA, USA), respectively. This identified the CASR deletion, but no other plausible candidates. The deletion segregated with hypocalcaemia (Fig. 1a). Despite normal bone density, in 2 5 years, our patient experienced 18 fractures of the ribs and feet – not classical sites for osteoporotic fracture. The cause of this high fracture rate is likely multifactorial – with reduced mobility, smoking and longterm prednisolone contributory. However, 16 fractures occurred in the year following initiation of risedronate (Fig. 1b). Whilst bisphosphonates prevent glucocorticoid-induced bone loss and fracture, we speculate that risedronate, in the milieu of low PTH and low bone turnover, which was further reduced by prednisolone, paradoxically increased fracture risk. The patient’s beta C-terminal telopeptide was undetectable on treatment and low in the proband (132 ng/l in III:2; Fig. 1a). As approximately 10% of adults with Langerhans’ cell histiocytosis have skeletal involvement, this is a potential aetiology of fractures in our patient. However, the presentation is typically with painful swelling of the affected site(s), and imaging shows a lytic lesion or associated soft tissue mass. As our patient had no clinical or radiological evidence of skeletal involvement, we hypothesize that bony Langerhans’ cell histiocytosis was not the cause of her recurrent fractures. Over 70 CASR mutations underlying autosomal dominant hypocalcaemia have been described. We report a novel amino acid deletion. Whilst functional studies may provide further evidence of pathogenicity, the data are already compelling. The deletion is in transmembrane domain 6 where causative mutations have been described (Fig. 1c). Functional studies report amino acids in this domain are critical for maintaining an inactive configuration. The

Exenatide corrects postprandial hyperglycaemia in young people with cystic fibrosis and impaired glucose tolerance: A randomized crossover trial

Impaired glucose tolerance (IGT) in cystic fibrosis (CF) manifests as postprandial hyperglycaemia. Pancreatic enzyme supplementation reduces the latter; restoring incretin secretion and slowing gastric emptying. We aimed to determine the acute effect of exenatide on postprandial glycaemia in young people with CF and IGT. Six participants with CF and IGT were studied on 2 days, in a double‐blind randomized crossover trial. After overnight fasting, they received exenatide 2.5 mcg or placebo (0.9% saline) subcutaneously 15 minutes before a pancake meal labelled with 13C octanoate and pancreatic enzyme replacement. The primary outcomes, area under the curve over 240 minutes (AUC 240) for blood glucose (P < 0.0001) and peak blood glucose (7.65 mM ± 0.34 [mean ± SE] vs 9.53 mM ± 0.63, P < 0.0001), were markedly lower after exenatide than placebo. AUC240 for insulin, C‐peptide, glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) was also lower after exenatide. Gastric emptying was markedly slower after exenatide, as assessed by time for 10% gastric emptying and peak 13CO2 excretion. We report for the first time that exenatide corrects postprandial hyperglycaemia in young people with CF and IGT. GLP‐1 agonists are a candidate treatment in CF‐related diabetes.