Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability.

Higher resolution whole-genome arrays facilitate the identification of smaller copy number variations (CNVs) and their integral genes contributing to autism and/or intellectual disability (ASD/ID). Our study describes the use of one of the highest resolution arrays, the Affymetrix(®) Cytogenetics 2.7M array, coupled with quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for detection and validation of small CNVs. We studied 82 subjects with ASD and ID in total (30 in the validation and 52 in the application cohort) and detected putatively pathogenic CNVs in 6/52 cases from the application cohort. This included a 130-kb maternal duplication spanning exons 64-79 of the DMD gene which was found in a 3-year-old boy manifesting autism and mild neuromotor delays. Other pathogenic CNVs involved 4p14, 12q24.31, 14q32.31, 15q13.2-13.3, and 17p13.3. We established the optimal experimental conditions which, when applied to select small CNVs for QMPSF confirmation, reduced the false positive rate from 60% to 25%. Our work suggests that selection of small CNVs based on the function of integral genes, followed by review of array experimental parameters resulting in highest confirmation rate using multiplex PCR, may enhance the usefulness of higher resolution platforms for ASD and ID gene discovery.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS: Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS: There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS: This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Outcome of array CGH analysis for 255 subjects with intellectual disability and search for candidate genes using bioinformatics.

Array CGH enables the detection of pathogenic copy number variants (CNVs) in 5-15% of individuals with intellectual disability (ID), making it a promising tool for uncovering ID candidate genes. However, most CNVs encompass multiple genes, making it difficult to identify key disease gene(s) underlying ID etiology. Using array CGH we identified 47 previously unreported unique CNVs in 45/255 probands. We prioritized ID candidate genes using five bioinformatic gene prioritization web tools. Gene priority lists were created by comparing integral genes from each CNV from our ID cohort with sets of training genes specific either to ID or randomly selected. Our findings suggest that different training sets alter gene prioritization only moderately; however, only the ID gene training set resulted in significant enrichment of genes with nervous system function (19%) in prioritized versus non-prioritized genes from the same de novo CNVs (7%, p < 0.05). This enrichment further increased to 31% when the five web tools were used in concert and included genes within mitogen-activated protein kinase (MAPK) and neuroactive ligand-receptor interaction pathways. Gene prioritization web tools enrich for genes with relevant function in ID and more readily facilitate the selection of ID candidate genes for functional studies, particularly for large CNVs.

Genomic changes detected by array CGH in human embryos with developmental defects.

Developmental abnormalities of human embryos can be visualized in utero using embryoscopy. Our previous embryoscopic and genetic evaluations detected developmental abnormalities in the majority of both euploid (74%) and aneuploid or polyploid (90%) miscarriages. Since we found the pattern of morphological changes to be similar in euploid and non-euploid embryos, we proposed that lethal submicroscopic changes, not detected by standard chromosome testing, may be responsible for miscarriage of euploid embryos. Whole genome oligo and bacterial artificial chromosome array comparative genome hybridization (CGH) was used to screen for submicroscopic chromosomal changes (DNA copy number variants or CNVs) in 17 euploid embryonic miscarriages, with a range of developmental abnormalities documented by embryoscopy. The CNV breakpoints were refined using a custom array (Agilent) with high resolution coverage of the CNVs. Six unique CNVs, previously not reported, were identified in 5 of the 17 embryos (29% of all cases or 50% of cases studied with higher resolution arrays). All six unique CNVs were <250 kb in size. On the basis of parental array CGH analysis, a de novo origin of a CNV was determined for one embryo (at 13q32.1) and suspected for another (at 10p15.3). Three CNVs, at Xq28, 1q25.3 and 7p14.3, were inherited and a CNV at 17p13.1 was of unknown origin. The genes contained within these unique CNVs will be discussed, with specific reference to rearrangements of syntaxin and tryptophan-aspartic acid (WD) repeat genes. Our report describes for the first time, de novo and inherited unique CNVs in euploid human embryos with specific developmental defects.

Molecular cytogenetic investigation of two patients with Y chromosome rearrangements and intellectual disability.

We describe two males with intellectual disability (ID) and facial dysmorphism, both of whom have non-mosaic Y chromosome rearrangements resulting in deletions of large portions of the Y chromosome. Patient A, with ID, mild dysmorphism, speech delay, Duane anomaly of the eye, hypermetropia and conductive hearing loss, had two structurally rearranged Y chromosomes resulting in both p and q arm deletions in addition to a Yp duplication. Patient B, also with speech and language delay, developmental delay and short stature, had an interstitial deletion of Yq11.21-11.23. Array-CGH excluded the presence of additional submicroscopic rearrangements at the 1 Mb resolution level. A review of males with Y chromosome rearrangements and ID was performed. Our study provides a more detailed molecular cytogenetic assessment of Y rearrangements in individuals with ID than has been previously possible, and facilitates assessment and comparison of other individuals with a Y chromosome rearrangement.

Putatively benign copy number variants in subjects with idiopathic autism spectrum disorder and/or intellectual disability.

Putatively benign copy number variants (bCNVs) can be broadly defined as DNA copy number gains or losses that do not lead to a recognizable clinical phenotype. Detection of bCNVs in genomes of clinically healthy individuals is increasing with the widespread use of whole genome arrays of different resolutions and the use of sequence comparison methods. However, the role of bCNVs in human disease susceptibility and phenotype diversity is mostly unknown. In order to explore a potential role of bCNVs in the susceptibility to and/or pathogenesis of human neurodevelopmental disorders we examined the frequency and type of common bCNVs (detected in >/=2 independent control studies) amongst 221 subjects with an autism spectrum disorder (ASD) and/or intellectual disability (ID) in comparison to 40 controls using three array platforms of increasing resolution (Spectral Genomics (1 Mb), Agilent (0.03 Mb) and NimbleGen (0.01 Mb)). We determined that the number of bCNVs/subject, type and frequency of most common bCNVs were similar for both the test and control cohorts when the same array platform was used. The comparison of the 'load' of bCNVs (i.e. number/subject) to a standardized metric of phenotypic features (see de Vries et al., 2001) in 91 ASD subjects revealed that a phenotype score >/=4 is significantly more common (P < 0.05) in persons with an ASD having one or more bCNVs via 1 Mb array-CGH, whereas individuals without any recognizable bCNVs are significantly more likely to have a less complex phenotype and a score

Oxytocin stimulates glucagon and insulin secretion in fetal and neonatal sheep.

In adults of several species arginine vasopressin (AVP) and oxytocin (OT) stimulate pancreatic secretion of immunoreactive plasma glucagon (IRG). In fetal sheep AVP is an important stress hormone and may be simultaneously secreted with OT; however, their effects on IRG secretion are not known. We sought to determine if AVP and/or OT affected pancreatic IRG secretion in fetal and neonatal sheep. Either AVP or OT was infused for 30 min in chronically catheterized fetal and neonatal sheep, obtaining peripheral arterial and/or portal venous blood samples before; 10, 15, and 30 min during; and 15, 30, and 60 min after infusion for measurements of blood gases, hematocrit, IRG, immunoreactive plasma insulin (IRI) and plasma glucose. AVP did not affect IRG or IRI in fetal sheep (mean +/- SE, 133 +/- 1 days gestation), but small increases occurred in portal venous blood of lambs (2-49 days old). In contrast, OT (4.6 +/- 0.3 mU/min.kg; n = 12) increased fetal plasma IRG from 72 +/- 5 to 86 +/- 6 and 97 +/- 7 pg/ml (P less than 0.001) and IRI from 16 +/- 2 to 20 +/- 3 and 20 +/- 2 microU/ml (P less than 0.02) at 15 and 30 min, respectively; 157 +/- 11 microU OT/min.kg had no effect. In lambs (2-49 days old), 3.0 mU OT/min.kg increased arterial (n = 15) IRG from 139 +/- 19 to 367 +/- 43 and 483 +/- 76 pg/ml (P less than 0.01) and portal IRG (n = 8) from 167 +/- 39 to 341 +/- 72 and 502 +/- 148 pg/ml (P less than 0.01), respectively. Arterial and portal IRI also rose (P less than 0.01) from 36 +/- 4 to 82 +/- 12 and 105 +/- 32 microU/ml and from 29 +/- 5 to 65 +/- 13 and 51 +/- 7 microU/ml, respectively. Glucose was unchanged in all experiments. In fetal and neonatal sheep, AVP has minimal effects on IRG and IRI release. In contrast, OT increases both substantially; furthermore, there is a difference in fetal and neonatal responsiveness. OT may be important in modulating glucagon and insulin secretion during and after parturition.

A study of the skin flora of spinal cord injured patients.

The skin flora of 11 spinally-injured patients was compared to that of 11 healthy control subjects. The perinea, groins, penile shafts and urethras of the patients were heavily colonized by a range of multi-drug resistant Gram-negative bacilli. Observations on patients from admission for up to 25 days suggest that the Gram-negative bacilli start to colonize the skin 2-3 days after admission. Some species, e.g., Citrobacter diversus and Escherichia coli appear as transient organisms while others such as Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae seem to become stable skin residents. The relationship of the skin flora to the organisms causing urinary tract infections in these patients was studied.

Asymptomatic bacteriuria and hemoglobin A1.

We measured hemoglobin A1 (HbA1) and performed clean-catch urine cultures in 752 patients (411 men and 341 women) with non-insulin-dependent diabetes mellitus (NIDDM) attending an outpatient diabetes clinic. Prevalence of bacteriuria was significantly greater in diabetic women than in controls (9.1 vs. 5.0%, P less than .001) but not in diabetic men. Risk of bacteriuria was not related to level of HbA1 at the time of urine culture. However, mean duration of diabetes mellitus was significantly greater in diabetic women with bacteriuria than in those without infection (9.9 +/- 1.5 vs. 5.4 +/- 0.4 yr, P less than .025), and the prevalence of bacteriuria was significantly greater in patients with complications of long-standing diabetes mellitus than in those without complications (P less than .005).

Multiple central venous line occlusions following an error in parenteral nutrition formulation.

Multiple occlusions of central venous catheters occurred in four patients who were receiving total parenteral nutrition (TPN). Misformulation of bulk prepared TPN solution caused temperature-dependent precipitation of calcium phosphate within the catheter during infusion.

Evidence for participation of the neurohypophysial hormones in the hyperglucagonemic response to hemorrhage in the rat.

Conscious adult male rats bearing jugular cannulae were injected with either normal rabbit serum (NRS) or with serum containing antibodies to both oxytocin (OT) and arginine vasopressin (AVP). In the NRS-treated group, plasma levels of OT, AVP and immunoreactive glucagon (IRG) were significantly elevated 10 min after hemorrhage (2.3 ml/100 g body weight over 5 min) whereas hyperglucagonemia was not detected in the antiserum-treated group until 30 min posthemorrhage. In animals which were deprived of water during the experiment, plasma IRG in the antiserum-treated group reached only 40% of the levels in the NRS-treated controls. These results suggest that hemorrhage-induced elevations in circulating AVP and/or OT contribute to increased release of glucagon by the endocrine pancreas consistent with previous demonstration of glucagonotropic activity of synthetic neurohypophysial peptides.

Providencia stuartii: a search for its natural habitat.

A selective differential medium has been used to search for Providencia stuartii in sewage, sewage contaminated natural waters and the faeces and skin of a small population of healthy non-hospitalized males. Colonization of 12 male patients with long-term indwelling bladder catheters and the general environment of the spinal injury unit was also examined. Providencia stuartii was not isolated from the non-hospital samples, but colonization of the urine (two patients) faeces (five patients) and skin (eight patients) was observed. Apart from equipment that had been in contact with patients urine or skin there was no general contamination of the ward environment suggesting that colonized patients were the main reservoir of this multiply antibiotic-resistant nosocomial pathogen.

Corticotropin-releasing factor inhibits insulin release from perfused rat pancreas.

The ability of synthetic corticotropin-releasing factor (CRF) (rat) to influence hormone release from the endocrine pancreas of rats has been evaluated by means of perfusion in situ. Release of insulin and glucagon into the perfusate was measured in the presence and absence of CRF (0.1, 1, and 10 ng/ml) under conditions of normal and high glucose concentration (5.5 and 11 mM). Under both conditions, CRF (0.1, 1, and 10 ng/ml) induced a rapid, dose-dependent inhibition of insulin release followed by a remarkable postinhibitory rebound when exposure to CRF was discontinued. CRF had no significant effect on glucagon release when tested under these conditions. These results are reminiscent of noradrenergic inhibition of insulin release and together with evidence for the presence of CRF in the pancreas suggest that this peptide could function as a neuromodulatory transducer in addition to its role as a hypophysiotropic hormone perhaps contributing to the coordination of the overall endocrine response to stress.

Corticotropin-releasing factor: its action on the islets of Langerhans.

Synthetic rat and ovine CRF were tested in an in vitro isolated pancreatic islet system for their ability to influence insulin and glucagon release. Acute exposure of islets to both rat and ovine CRF resulted in a significant increase in glucagon release but only over a narrow range of concentration (50-200 pg/ml). Neither peptide had a significant effect on insulin release. Our results raise the possibility that release of glucagon may be stimulated by CRF as part of the overall response to stress.

Hypothalamic lesions in the weanling rat alter pancreatic response to glucose.

Circulating levels of insulin and glucagon were monitored daily in weanling rats bearing bilateral radiofrequency lesions of the hypothalamic region comprising the ventral pole of the dorsomedial nucleus and at least one third of the dorsal pole of the ventromedial nucleus (V-DMH). Plasma insulin levels in the animals with lesions were significantly elevated by the eighth post-lesion day while plasma glucagon levels were significantly reduced by the 13th day. An intravenous glucose bolus administered to conscious unrestrained animals with lesions had no significant effect on circulating insulin levels but resulted in a dramatic increase in circulating glucagon levels. The IV glucose injections had no significant effect on circulating glucagon levels in the sham-lesioned and unoperated controls while the plasma insulin levels in both control groups were significantly elevated. After a glucose challenge in vitro (300 mg%), insulin release by islets from the lesioned animals showed only a slight increase whereas glucagon release was paradoxically increased. These results provide evidence for an abnormal glucose-sensing function of the pancreatic islet after hypothalamic lesions.

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP).

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.

Actions of neurohypophysial peptides on pancreatic hormone release.

The intermittent reports concerning metabolic actions of neurohypophysial extracts or hormones encouraged us to study the effect of these substances on the function of the endocrine pancreas. A surprisingly small amount of neural lobe (NL) extract (0.025 NL eq/ml) stimulated a 425% increase in the release of glucagon from islets isolated from the pancreas of the rat. Gel filtration of the extract produced an elution profile of glucagon-releasing activity that was superimposable on the profiles of oxytocin (OT) and arginine vasopressin (AVP). Synthetic OT and AVP each elicited a concentration-dependent stimulation of glucagon release but failed to influence insulin release in medium 199 containing 5.6 mM glucose. They were effective at 0.2 ng/ml (+55%, +50%) and produced a striking increase (five- to sevenfold) at 20 ng/ml. The response to each peptide was greatly diminished in the presence of a higher concentration of glucose (11 mM). The lysine, desamino-, and 1,6-aminosuberyl analogues of vasopressin, vasotocin, and AVP are equipotent peptides, whereas the desglycinamide analogue, pressinoic acid, and angiotensin II were inactive. Injection of AVP (1 microgram iv) produced a rapid increase in peripheral glucagon (+185% in 5 min). The response to injection of OT was less rapid (+105% in 15 min), but in each case elevation of insulin was also observed. Our results provide evidence that OT and AVP can act directly on the endocrine pancreas and may help explain previous reports of metabolic actions of these peptides.

Purification of a bioactive FSH-releasing factor (FSHRF).

Before the advent of radioimmunoassay (RIA), FSH-releasing factor (FSHRF) appeared to be separable from LH-releasing hormone (LHRH) by chromatography followed by bioassay for FSH. In this study, we re-examined hypothalamic extracts for the existence of an FSHRF distinct from LHRH, utilizing the Steelman-Pohley bioassay as well as RIA for identification of FSH. Acid extracts of rat hypothalamic fragments were chromatographed on Sephadex G-25. LH- and FSH-releasing activities of each fraction were assessed by bio- and immunoassay of FSH and immunoassay of LH released after incubation with hemipituitaries from adult male rats. The immunoreactive LHRH(IR-LHRH) concentration of each fraction was also measured by RIA. In order to evaluate the FSH-releasing activity of LHRH, three doses of synthetic LHRH were tested and FSH-releasing activity determined by bio- and immunoassay. By RIA, the FSH-releasing activity of each column fraction could be accounted for by IR-LHRH contamination. However, greater FSH-releasing activity than could be predicted by IR-LRH contamination was detected by Steelman-Pohley assay in fractions eluted prior to the LHRH peak in 2 separate fractionations. These fractions from the second fractionation were pooled and eluted from a CMC column with ammonium acetate buffers. Again greater FSH-releasing activity than could be accounted for by IR-LHRH was detected prior to the IR-LHRH peak by Steelman-Pohley assay. These results agree with early work from our laboratory and suggest the presence of a bioactive FSHRF in hypothalamic extracts.

Purification of a glucagon releasing factor from the rat hypothalamus.

The purification of a peptide from the rat hypothalamus which is capable of stimulating the release of glucagon from the endocrine pancreas and therefore named glucagon releasing factor (GlRF), is described. Compositional analysis of the product obtained using well established techniques for peptide isolation revealed that GlRF appeared to consist of 30-31 amino acids. The significance of GlRF in the physiological regulation of glucagon secretion is yet to be determined but the existence of this very potent factor supports the concept of a hypothalamic-pancreatic neurohormonal link.

Failure of coagulase-negative staphylococci to transfer antibiotic resistance to Staphylococcus aureus 1030 in mixed cultures.

One hundred and fifty-three isolates of coagulase-negative staphylococci obtained from human skin failed to transfer resistance to either cadmium ions (46 isolates), trimethoprim (37 isolates), erythromycin (25 isolates) or tetracycline (45 isolates) to Staphylococcus aureus strain 1030 or to each of 10 of its lysogenic derivatives in mixed cultures. Thirty-three trimethoprim-resistant coagulase-negative staphylococcal isolates obtained from the human intestine also failed to transfer this resistance to the recipients in mixed cultures.