Martin McMillan

Intrafamilial Genotyping of Helicobacter pylori from Faecal DNA.

Helicobacter pylori infection, often acquired in early childhood, is a global cause of undernutrition, gastritis, peptic ulcer disease and gastric carcinoma. This study tested the feasibility of using H. pylori shed in the faeces as a source of DNA for non-invasive epidemiological studies. H. pylori DNA was chemically recovered and isolated using a specific biotinylated oligonucleotide probe with magnetic capture from 28 H. pylori positive faecal samples obtained from children attending hospital for the investigation of suspected H. pylori infection, together with close family members. Random amplification of polymorphic DNA (RAPD) was subsequently used to discriminate each isolate. 93% of stool samples selected were typeable. Parent, child and sibling samples were compared and similarities determined. Phylogenetic analysis showed that H. pylori DNA obtained from the faeces can be used to genotype individual strains, offering a means of studying intrafamilial transfer of this microorganism.

Evaluation of Protocol Using Gene Capture and PCR for Detection of Helicobacter pylori DNA in Feces

ABSTRACT The route of transmission of Helicobacter pylori, which is usually acquired in childhood and is one of the most common bacterial infections in humans, remains undetermined. Mapping the distribution of H. pylori genotypes within families could help to determine the routes of transmission and risk factors. Here we describe a noninvasive method for obtaining H. pylori DNA isolates from the feces of children. Children presenting with gastrointestinal symptoms at the Royal Hospital for Sick Children were tested for gastric H. pylori colonization by using the 13C-urea breath test (UBT) and were asked to provide fecal samples, which were tested for H. pylori by using the HpSA fecal antigen test. DNA was purified from fecal samples by using a novel method of gene capture with subsequent H. pylori PCR analysis. Fifteen UBT-positive and 15 UBT-negative children participated in the study. The positive and negative predictive values for the assay were 80 and 100%, respectively. Fecal DNA purification followed by H. pylori PCR analysis is an effective tool for harvesting H. pylori DNA isolates from the feces of children. This technique may be developed to allow the diagnosis and noninvasive genotyping of H. pylori in children and their families.

Deficits in Trabecular Bone Microarchitecture in Young Women With Type 1 Diabetes Mellitus

The pathophysiological mechanism of increased fractures in young adults with type 1 diabetes mellitus (T1DM) is unclear. We conducted a case‐control study of trabecular bone microarchitecture and vertebral marrow adiposity in young women with T1DM. Thirty women with T1DM with a median age (range) age of 22.0 years (16.9, 36.1) attending one outpatient clinic with a median age at diagnosis of 9.7 years (0.46, 14.8) were compared with 28 age‐matched healthy women who acted as controls. Measurements included MRI‐based assessment of proximal tibial bone volume/total volume (appBV/TV), trabecular separation (appTb.Sp), vertebral bone marrow adiposity (BMA), and abdominal adipose tissue and biochemical markers of GH/IGF‐1 axis (IGF‐1, IGFBP3, ALS) and bone turnover. Median appBV/TV in cases and controls was 0.3 (0.22, 0.37) and 0.33 (0.26, 0.4), respectively (p = 0.018) and median appTb.Sp in T1DM was 2.59 (2.24, 3.38) and 2.32 (2.03, 2.97), respectively (p = 0.012). The median appBV/TV was 0.28 (0.22, 0.33) in those cases with retinopathy (n = 15) compared with 0.33 (0.25, 0.37) in those without retinopathy (p = 0.02). Although median visceral adipose tissue in cases was higher than in controls at 5733 mm3 (2030, 11,144) and 3460 mm3 (1808, 6832), respectively (p = 0.012), there was no difference in median BMA, which was 31.1% (9.9, 59.9) and 26.3% (8.5, 49.8) in cases and controls, respectively (p = 0.2). Serum IGF‐1 and ALS were also lower in cases, and the latter showed an inverse association to appTbSp (r = –0.30, p = 0.04). Detailed MRI studies in young women with childhood‐onset T1DM have shown clear deficits in trabecular microarchitecture of the tibia. Underlying pathophysiological mechanisms may include a microvasculopathy.

Prevalence of Helicobacter pylori vacA, cagA and iceA genotypes in South African patients with upper gastrointestinal diseases.

Clinical response to Helicobacter pylori infection may be determined by specific virulence-associated genotypes which varies geographically. The aim of this study was to investigate the diversity of putative virulence markers of H. pylori; cagA, vacA and iceA in the Eastern Cape Province of South Africa. One hundred H. pylori strains obtained from dyspeptic patients were used. Gastric biopsies were obtained from 254 dyspeptic patients. H. pylori was cultured and strains were studied. Bacterial genotypes cagA, vacA (s and m subtypes) and iceA were analysed by PCR using specific primers. CagA was identified in 90% of the strains investigated. Fifty-eight of the 100 strains had the vacA signal sequence genotype s1 and 26 had subtype s2. Combined vacA s1/s2 was detected in 16 of the strains. VacA middle region analysis showed that 8 (8%) strains were m1 while 50 were m2. Combined vacA m1/m2 was detected in 36 of the strains. s1m2 (20%) and s2m2 (20%) genotypes were the most common allelic combinations of the vacA gene among the strains. Multiple vacA genotypes were detected in this study. Twenty-six percent of the strains identified had both iceA1 and iceA2. All our strains tested positive for the ureC (glmM) gene. This study reveals a high prevalence of vacA, cagA and iceA2.

A Prospective Longitudinal Study of Growth and Pubertal Progress in Adolescents with Inflammatory Bowel Disease

Background: Puberty and growth may be affected in inflammatory bowel disease (IBD) but the extent is unclear. Methods: We performed a prospective study over 12 months in 63 adolescents (Crohns disease, CD, n = 45; ulcerative colitis/IBD unclassified, UC, n = 18) with a median age of 13.4 years (range 10-16.6). Assessment included anthropometry, biochemical markers of growth and puberty and an assessment of quality of life by IMPACT-III. Results: Compared to the normal population, boys with CD were shorter, with a median height SDS (HtSDS) of -0.13 (-2.52 to 1.58; p < 0.05). In addition, the study cohort had a lower median IGF-1 SDS of -0.29 (-4.53 to 2.96; p = 0.008) and a higher median IGFBP3 SDS of 0.45 (-3.15 to 2.55; p = 0.002). Over the study period, the median Ht velocity (HV) was 5 cm/year (0.2-8.7) and the change in HtSDS was 0.06 (-0.48 to 0.57). The median difference between the chronological and bone age was 0.3 years (-2.5 to 3.0) and pubertal examination was not delayed. In the whole group, the erythrocyte sedimentation rate (ESR) showed an inverse association with HV (r = -0.29; p = 0.025) and IGF-1:IGFBP3 (r = -0.34; p = 0.016). The score in the body image domain, IMPACT-III, was inversely associated with HtSDS (r = -0.31; p = 0.03). Conclusion: Despite no evidence of pubertal delay, adolescents with IBD display growth retardation which may be associated with raised ESR, adverse quality of life measures and an abnormality of IGF-1 bioavailability.

Prevalence of endocrine and genetic abnormalities in boys evaluated systematically for a disorder of sex development

Abstract STUDY QUESTION What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)? SUMMARY ANSWER An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases. WHAT IS KNOWN ALREADY Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear. STUDY, DESIGN, SIZE, DURATION This study was a retrospective review of investigations performed on 122 boys. PARTICIPANTS/MATERIALS, SETTING, METHODS All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1–11). Details of phenotype, endocrine and genetic investigations were obtained from case records. MAIN RESULTS AND THE ROLE OF CHANCE An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1–10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5–11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5–11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1–9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations. LIMITATIONS, REASONS FOR CAUTION A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel. WIDER IMPLICATIONS OF THE FINDINGS The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD. STUDY FUNDING/COMPETING INTEREST(S) RN was supported by the James Paterson Bursary and the Glasgow Childrens Hospital Charity Summer Scholarship. SFA, RM and EST are supported by a Scottish Executive Health Department grant 74250/1 for the Scottish Genomes Partnership. EST is also supported by MRC/EPSRC Molecular Pathology Node and Wellcome Trust ISSF funding. There are no conflicts of interest. TRIAL REGISTRATION NUMBER None.

Androgen-responsive non-coding small RNAs extend the potential of HCG stimulation to act as a bioassay of androgen sufficiency

BACKGROUND It is unclear whether a short-term change in circulating androgens is associated with changes in the transcriptome of the peripheral blood mononuclear cells (PBMC). AIMS AND METHODS To explore the effect of hCG stimulation on the PBMC transcriptome, 12 boys with a median age (range) of 0.7 years (0.3, 11.2) who received intramuscular hCG 1500u on 3 consecutive days as part of their investigations underwent transcriptomic array analysis on RNA extracted from peripheral blood mononuclear cells before and after hCG stimulation. RESULTS Median pre- and post-hCG testosterone for the overall group was 0.7 nmol/L (<0.5, 6) and 7.9 nmol/L (<0.5, 31.5), respectively. Of the 12 boys, 3 (25%) did not respond to hCG stimulation with a pre and post median serum testosterone of <0.5 nmol/L and <0.5 nmol/L, respectively. When corrected for gene expression changes in the non-responders to exclude hCG effects, all 9 of the hCG responders consistently demonstrated a 20% or greater increase in the expression of piR-37153 and piR-39248, non-coding PIWI-interacting RNAs (piRNAs). In addition, of the 9 responders, 8, 6 and 4 demonstrated a 30, 40 and 50% rise, respectively, in a total of 2 further piRNAs. In addition, 3 of the responders showed a 50% or greater rise in the expression of another small RNA, SNORD5. On comparing fold-change in serum testosterone with fold-change in the above transcripts, a positive correlation was detected for SNORD5 (P = 0.01). CONCLUSIONS The identification of a dynamic and androgen-responsive PBMC transcriptome extends the potential value of the hCG test for the assessment of androgen sufficiency.

An Unbalanced Rearrangement of Chromosomes 4:20 is Associated with Childhood Osteoporosis and Reduced Caspase-3 Levels.

The purpose of this study was to investigate the association of a chromosome 4:20 imbalance with osteoporosis in three related children. Bone biochemistry, bone turnover markers, and dual-energy X-ray absorptiometry (DXA) scanning were performed in all three cases and bone biopsy and histomorphometry in one. The chromosome imbalance was delineated by array comparative genomic hybridization (aCGH) and analyzed for candidate genes. A potential candidate gene within the deleted region is caspase-3, previously linked to low bone mineral density (BMD) in heterozygous mice thus caspase-3 activity was measured in cases and controls. Routine bone biochemistry and markers of bone turnover did not reveal any abnormality. DXA showed reduced total and lumbar spine bone mineral content. aCGH showed an 8 megabase (Mb) deletion of terminal chromosome 4q incorporating a region previously linked to low BMD and a 15 Mb duplication of terminal chromosome 20p. Bone biopsy showed a high bone turnover state, trabecularisation of cortical bone and numerous small osteoclasts coupled with normal bone formation. Basal serum caspase-3 activity was lower in cases compared with controls. We conclude that the early-onset osteoporosis with low basal levels of caspase-3 and abnormal osteoclasts is a feature of this chromosomal translocation. Further investigation of the role of the deleted and duplicated genes and especially caspase-3 is required.

G94 Characterising Changes in the in Vivo Rodent Brain Using Magnetic Resonance Spectroscopy

Background By providing a non-invasive, functional insight, Magnetic Resonance Spectroscopy (MRS) has the potential to provide objective longitudinal data on mammalian brain development. Aim To assess the sexual dimorphism in rodent brain chemistry and development using in vivo MRS. Methods 26(19 male) Sprague-Dawley rats were scanned at 6wks and 20(16 male) at 10wks using a 7TMRI scanner. Testosterone concentrations were measured by ELISA. Metabolites were expressed as a ratio to creatine and full width at half-maximum (FWHM) of the water peak was used as a guide to the reliability of the ratios. Results Median weight in 6wk males (M6) and females (F6), 10wk males (M10) and females (F10) was 197g(range,142–230), 131g (121–135), 316g(274–365) and 206g(191–210) respectively. Median anogenital distance (AGD) in M6, F6, M10, F10 was 2.46cm (1.89–2.9), 1.17cm(1.04–1.19), 3.25cm(2.8–3.6) and 1.33cm(1.07–1.60). Median serum testosterone in M6 and M10 were 1.33ng/ml (0.23–5.45) and 3.36ng/ml(1.75–8.26). 14 metabolites were identified in the occipitofrontal cortex. FWHM range was within the optimal range at 12–38Hz. In M6, myo-inositol ratios showed a positive association with circulating testosterone (p = 0.04), and AGD was correlated with phosphocreatine (p = 0.033) and glutamate (p = 0.045). There was a difference between M6 and F6 in 3 metabolite ratios: phosphocholine (p = 0.014), lactate (p = 0.046) and NAA (p = 0.005). In addition, in males, there was an increase from 6wks to 10wks in 3 metabolite ratios: taurine (p = 0.025), myo-inositol (p = 0.012) and phosphocholine (p = 0.005). Conclusions MRS is a reliable tool for studying the brain in maturing rats and may be a useful tool for studying the link between longitudinal changes in sex steroids and brain development.