Polonis, Katarzyna

Abstract Background Massively parallel sequencing (MPS) of nucleic acids has been a transformative technology for basic and applied genomic science, increasing efficiencies and decreasing costs to enable studies of unprecedented scope and impact. In clinical settings, these technological and scientific advances have led to the development of tests that are increasingly fast, comprehensive, and more frequently employed. Practitioners of genomic medicine have applied these tools across clinical settings, including diagnosis of inherited disorders and cancers and infectious disease detection and surveillance. In recent years, the commercial marketplace for MPS sequencers and reagents has been dominated by a few companies. The growing demand for sequencing has led to the recent emergence of several new sequencing platforms with techniques that may provide alternatives or improvements to existing workflows or allow the adoption of sequencing workflows in new settings. Clinical genomics laboratories will evaluate these platforms from a unique perspective, focusing on how technological advancements can improve patient care. Content This review describes short-read sequencing platforms provided by Illumina, Element Biosciences, MGI, PacBio, Singular Genomics, Thermo Fisher Scientific, and Ultima Genomics. This review discusses their innovative approaches, principles, workflows, and applications. Summary This review aims to inform laboratory geneticists, clinicians, and researchers about emerging short-read technologies and their applications in clinical genomics. By highlighting their principles and potential contributions, we aim to assist laboratories in selecting suitable solutions for their sequencing needs considering key factors such as applications, throughput, and integration with existing laboratory workflows.

AbstractAxenfeld‐Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Background Caveolin‐1 is a structural protein of caveolae which forms a signaling platform for several cell surface receptors including leptin and insulin receptor. Caveolin‐1 expression is increased with obesity which is implicated in impaired adipose tissue leptin signaling. Considering the overlap between insulin and leptin cellular signaling pathways, we investigated known insulin‐sensitizing drugs which may reduce caveolin‐1 expression in adipose tissue and consequently improve leptin signaling. Methods We used an in‐vitro approach to identify insulin‐sensitizing drugs which may reduce caveolin‐1 expression. Differentiated human white preadipocytes (dHWP) were treated with pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 expression was determined by Western blot. We further tested the responses in human adipose tissue from obese individuals (n=12, age: 45.4 ± 9.2 years, BMI: 48.9 ± 7.3 kg/m 2 ) undergoing surgery. Tissue was incubated with drugs for 24 h and caveolin‐1 expression was determined. Furthermore, the effect of drugs on leptin‐dependent activation of ERK and STAT3 was examined by western blot. Results Among the drugs investigated, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 protein (p=0.01) expression in dHWP. Treatment with increasing concentrations of troglitazone upregulated caveolin‐1 (p=0.01), while pioglitazone (p=0.50) and glimepiride (p=0.09) had no effect of caveolin‐1 protein expression. Given these findings, only metformin was tested in human adipose tissue. Overall, metformin did not alter caveolin‐1 expression in adipose tissue from obese individuals (n=12, control: 0.040 ± 0.056, metformin: 0.024 ± 0.030, p=0.57), although a metformin dependent decrease in caveolin‐1 was evident in adipose tissue from 7 of the 12 obese subjects (control: 0.062 ± 0.066, metformin: 0.014 ± 0.013, p=0.02). Notably, changes in caveolin‐1 expression induced by metformin treatment were positively correlated with BMI (p=0.05, r 2 =0.33, n=12). Also, leptin dependent activation of ERK (control: 0.26 ± .098, leptin: 0.28 ± .08) and STAT3 (control: 0.028 ± 0.027, leptin: 0.032 ± 0.023) was impaired in obese adipose tissue and did not change with metformin treatment even in the 7 individuals with metformin associated decreases in caveolin‐1. Conclusions Metformin reduces caveolin‐1 expression in differentiated HWP. However, metformin decreases caveolin‐1 expression in adipose tissue of some but not all obese subjects, and does not have any effects on leptin signaling. The heterogeneous effects of metformin on caveolin‐1 expression in adipose tissue from obese individuals may be related to BMI and needs further investigation. Support or Funding Information MAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01. PS was supported by AHA scientist development grant 11SDG7260046.

Background Obesity‐associated metabolic disorders have been shown to be related to body fat distribution. Specifically, increases in visceral fat are more detrimental than increases in subcutaneous fat depots. However, the underlying mechanisms through which visceral fat potentiates cardiometabolic dysregulation are not completely understood. In recent years, the presence of localized renin angiotensin system (RAS) in adipose tissue and its role in regulating blood pressure and metabolism has been recognized. Therefore, we investigated if differential expression of RAS components in visceral versus subcutaneous adipose tissue may help explain pathological effects of visceral fat. Hypothesis We hypothesized that compared to subcutaneous adipose tissue, visceral adipose tissue has increased expression of RAS proteins involved in the pro‐inflammatory and pro‐fibrotic ANGII‐AT1R pathway but attenuated expression of proteins mediating the anti‐inflammatory and anti‐fibrotic ANG1‐7‐MASR pathway. Methods We determined the expression of RAS proteins in paired subcutaneous and visceral (omental) adipose tissue samples from obese individuals undergoing surgery (n = 30, BMI: 46.1 ± 7.3 kg/m 2 , age: 46.5 ± 14.4 years) by Western blot. We examined proteins involved in 1) Ang II generation: angiotensinogen (AGT), angiotensin converting enzyme (ACE), chymase; 2) Ang1‐7 generation: ACE‐2; and 3) cellular receptors: angiotensin I receptor (AT1R), MAS receptor (MASR), and aldosterone receptor. Results Comparison of protein expression between subcutaneous and visceral adipose tissue samples showed no changes in proteins involved in generation of angiotensin peptides, namely AGT (0.64 ± 0.57 vs. 0.33 ± 0.28, p = 0.11, n = 12), chymase (0.27 ± 0.23 vs. 0.36 ± 0.41, p = 0.77, n = 22), and ACE‐2 (0.23 ± 0.41 vs. 0.08 ± 0.09, p = 0.21, n = 15). However, ACE expression tended to be higher in subcutaneous vs. visceral fat (0.10 ± 0.06 vs. 0.06 ± 0.03, p = 0.052, n = 12). Furthermore, expression of cellular receptor AT1R was higher in subcutaneous fat (0.24 ± 0.34 vs. 0.07 ± 0.09, p = 0.03, n = 12), and MASR was lower in subcutaneous fat (0.22 ± 0.26 vs. 0.88 ± 0.99, p = 0.01, n = 20). Aldosterone receptor expression (0.21 ± 0.22 vs. 0.14 ± 0.13, p = 0.52, n = 11) was not altered in subcutaneous and visceral depots. Conclusions Proteins involved in the generation of angiotensin peptides are not differentially expressed in subcutaneous versus visceral fat depots. However, expression of cellular receptors is different such that inflammatory and fibrotic effects of RAS may be more predominant in subcutaneous fat. Therefore, RAS is likely to play a role in limiting subcutaneous fat expansion thereby leading to increased visceral fat depots. Furthermore, our findings do not support the role of visceral RAS in development and progression of cardiovascular pathology associated with increased visceral fat depots. Support or Funding Information MAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01.

Background Obstructive sleep apnea (OSA) is characterized by repetitive intermittent hypoxia and reoxygenation during sleep, and is a risk factor for cardiometabolic diseases including hypertension and diabetes. The renin‐angiotensin system (RAS) is an important cardiovascular and metabolic regulator playing key roles via angiotensin peptides. Recent studies demonstrate that all components of the RAS, including enzymes responsible for AngII generation, such as angiotensin converting enzyme (ACE) and chymase, are present in adipose tissue. Furthermore adipose tissue significantly contributes to systemic levels of AngII, thereby potentiating development of hypertension in obesity. Given that OSA patients are mostly obese, the localized effects of RAS in adipose tissue are likely an important mediator of OSA‐related hypertension. This study sought to investigate the effects of intermittent hypoxia (IH), a hallmark of OSA, on Ang II generation pathways in preadipocytes. Methods/Results RAS plays an important role in adipose tissue via its action on preadipocytes. Therefore, we examined the effects of IH in primary cultures of human white preadipocytes (HWP). HWP cells were exposed to repeated cycles of 30 min‐21% O 2 (normoxic) and 30 min‐1% O 2 (hypoxic) at 37°C for 24 hours. Cells grown in continuous normoxic conditions were used as controls. mRNA and protein expression of ACE, chymase, renin, AT1, and AT2 receptors were investigated by semi‐quantitative RT‐PCR and Western blot, respectively. Ang II secretion was examined by enzyme‐linked immunosorbent assay in condition medium. Results IH upregulated ACE (P=0.04) and chymase (P=0.05) expression while the expression of renin (P=0.94), AT1R (P=0.30), and AT2R (P=0.58) receptors did not change in HWP. Additionally, IH increased Ang II secretion in conditioned media (P=0.01). Conclusions This is the first study to demonstrate intermittent hypoxia‐mediated upregulation of Ang II generating enzymes ACE and chymase, along with increased AngII secretion in HWP. Together, our data strongly support the role of IH in upregulating RAS via increases in AngII. These studies suggest a role for adipose tissue and RAS in OSA related pathophysiology. Support or Funding Information NIH R01 grant (#HL65176); Becari C. is supported by grant from CNPq‐Brazil (# 203802/2014‐4).

BACKGROUND Leptin is an adipokine with a central role in energy homeostasis and appetite regulation. Increases in leptin suppress appetite, while decreases in leptin increase appetite and may consequently cause weight gain. Patients with established obstructive sleep apnea (OSA) have been shown to have elevated leptin. Also, newly diagnosed OSA patients have a history of significant recent weight gain. Repetitive nocturnal intermittent hypoxia has been implicated in OSA pathophysiology and in increases in leptin. However, the factors contributing to weight gain in OSA are not completely understood. In contrast to the history of weight gain in OSA subjects, animal models of intermittent hypoxia (IH) are associated with weight loss along with increases in leptin. Furthermore, acute altitude dependent hypoxia in humans has also been associated with loss of appetite and increases in leptin. We postulated that the duration of IH may be important in defining the nature of effects on leptin. In this study we sought to examine the interactions between IH and leptin secretion, comparing acute versus chronic exposure to IH. We tested the hypothesis that although acute IH increases leptin secretion and likely suppresses appetite, repeated chronic exposure to IH will decrease leptin secretion, and thereby may increase appetite and predispose individuals such as patients with OSA to weight gain. METHODS We used an in‐vitro approach to examine the acute effects of IH (24 cycles of 30 min 0.1% O 2 followed by 30 min exposure to 21% O 2 ) in differentiated human white preadipocytes. Cells grown in normoxic conditions and continuous hypoxia were controls for these experiments. For chronic treatment, cells were exposed to 9 cycles of IH each day for 7 days. Leptin secretion was measured in 48 h conditioned media by ELISA. RESULTS Acute 24 hour exposure to IH or sustained hypoxia increased leptin secretion (p = 0.013). However, chronic 7 day exposure of differentiated human white preadipocytes caused decreases in leptin secretion (p = 0.02). CONCLUSION Acute intermittent hypoxia elicits increases in leptin secretion. Conversely, chronic IH decreases leptin secretion, suggesting a mechanism for development of obesity in the early stages of OSA. We speculate that decreased leptin, as a consequence of chronic IH, would result in increased appetite and hence weight gain. As obesity progresses, leptin levels likely rise secondary to the increase in body fat. Elevated leptin levels in patients with longstanding OSA may be indicative of increased fat mass and not a consequence of IH‐mediated effects on adipocytes. Support or Funding Information Christiane Becari was supported by a grant from CNPq Brazil (# 203802/2014‐4); Prachi Singh was supported by AHA scientist development grant (11SDG7260046).

Background Obstructive sleep apnea (OSA) patients have elevated levels of plasma leptin and are exposed to repeated nocturnal intermittent hypoxia (IH). Leptin induces increased adiponectin expression, suggesting that adiponectin should be increased in OSA. However, levels of plasma adiponectin, an anti‐atherogenic and insulin‐sensitizing adipokine, are inconsistent in OSA, and adiponectin levels are often low. Both increases in leptin and decreases in adiponectin may contribute to increased cardiometabolic risk in OSA. In this study we sought to examine the interactions between IH, leptin, and adiponectin. Using an in‐vitro approach in differentiated human white preadipocytes (dHWP), we tested the hypothesis that adiponectin expression in response to acute IH‐mediated increases in leptin may be modulated in the presence of hypoxia. Methods dHWP were exposed to 24 cycles of IH (30 min 0.1% O2 followed by 30 min exposure to 21% O2), continuous hypoxia (0.1% O2) or continuous normoxia (21% O2, control). Secretion of leptin and adiponectin in the 24h conditioned media was determined by ELISA and intracellular changes in adiponectin were determined by Western blot analysis. Results We show that acute 24 hour exposure to IH or sustained hypoxia increased leptin secretion (p = 0.013) but did not alter adiponectin secretion (p = 0.48). In fact, the intracellular expression of adiponectin actually decreased during the acute exposure to both IH and to continuous hypoxia (p = 0.02). Also, while leptin increased intracellular adiponectin expression in normoxic conditions (p = 0.03), it was unable to do so in the presence of IH and hypoxia (p = 0.36). Conclusions Acute IH and continuous hypoxia mediate increases in leptin, but not in adiponectin secretion. Furthermore, although leptin increases adiponectin in normoxic conditions, IH and continuous hypoxia cause decreases in intra‐cellular adiponectin expression and also attenuate leptin‐mediated increases in adiponectin expression. These hypoxia‐effects on adiponectin expression may help explain the low adiponectin levels in OSA, which may contribute to increased cardiovascular and diabetes risk. Support or Funding Information Christiane Becari was supported by a grant from CNPq Brazil (# 203802/2014‐4); Michaela Pfeifer was supported by the APS STRIDE summer undergraduate research fellowship (NHLBI 1R25HL115473‐01); Prachi Singh was supported by AHA scientist development grant 11SDG7260046.

Background Obstructive sleep apnea (OSA) increases the risk for metabolic and cardiovascular disease. Telomere shortening has been linked to hypertension, diabetes mellitus, and cardiovascular diseases such as stroke and myocardial infarction. Because these conditions are highly prevalent in sleep apnea, we hypothesized that telomere length (TL) would be reduced in patients with OSA. Methods Genomic DNA was isolated from circulating leukocytes in 176 subjects (42.8 ± 13.8 years, 22.7 % women) who had undergone overnight polysomnographic evaluation. Of these individuals, 92 subjects had an Apnea Hypopnea Index (AHI) >5 events/hour and were classified as OSA patients. TL was measured using a quantitative PCR where a telomere to single copy gene ratio is determined and represented as number of base pairs (bp). The effect of OSA on TL was determined by comparing a control group (no OSA) (AHI<5, n=92) with patients with mild OSA (≥ 5 AHI <15, n=25), and those with moderate to severe OSA (AHI ≥15, n=59) in unadjusted and age‐adjusted models. Results Compared to control subjects, mild and moderate to severe OSA subjects were older: 48.9 ± 11.2 vs. 36.0 ± 11.2 years (p<0.001) and 50.8 ± 13.1 vs. 36.0 ± 11.2 years (p<0.001), respectively. Statistical models examining the effects of OSA severity on TL showed a J‐shaped curve : longer TL in moderate to severe OSA (4867 ± 221 bp) and controls (4820 ± 160 bp), and the shortest TL in mild OSA (4742 ± 155 bp). The mean TL in moderate to severe OSA was significantly longer than in mild OSA (p=0.0048). These effects were more pronounced after adjustment for age, when we observed significant differences in mean TL between moderate to severe OSA and mild OSA (p=0.0036) and between moderate to severe OSA and the control group (p=0.0379) (). However, the mean TL between control subjects and subjects with mild OSA was not different. Conclusion Moderate to severe OSA patients had longer TL compared with mild OSA and control group. This association was independent of age. Even though OSA is associated with increased cardiovascular risk, telomere lengthening in subjects with moderate to severe OSA suggests the presence of protective compensatory mechanisms to prevent telomere shortening in OSA. On the other hand, the existence of longer telomere in subjects with moderate and severe OSA may also indicate triggering of molecular mechanisms which may contribute to cancer development. Further studies are needed to explain the role of hypoxia in telomere dynamics in OSA, and the interactions between telomere length and increased longevity reported in older patients with OSA. Support or Funding Information NIH R01 grant (#HL65176); Singh P. is supported by AHA scientist development grant (11SDG7260046). Becari C. is supported by grant from CNPq‐Brazil (# 203802/2014‐4).

Introduction: Renin-Angiotensin System (RAS) regulates the cardiovascular system and has been implicated in the development of abdominal aortic aneurysm (AAA). RAS modulates function via balancing the ACE/Chymase/AngII-AT1 receptor and ACE-2/Ang(1-7)-Mas receptor axis. Preferential activation of the ACE/Chymase/AngII-AT1 receptor pathway of the RAS contributes to dysregulation of cardiovascular system and disease while the Ang(1-7)-MasR axis contributes to anti-inflammatory pathways. Therefore, identifying the RAS axis mechanism involved in growth, progression, or the risk of rupture in AAA humans could bring a novel treatment perspective. Hypothesis: We hypothesize that in the AAA, the local RAS is altered such as it favours the ACE/AngII/AT1R axis while decreasing the ACE-2/Ang(1-7)/MasR pathway, with consequent AAA development. Objective: We sought to investigate the expression of RAS components in the AAA human aorta. Methods: Aorta tissue from AAA patients (n=21) was collected during the AAA conventional repair surgery, and aorta control aortas were obtained from organ donors (Control, n=11). Protein expression of renin, chymase, ACE, ACE-2 AT1, AT2, and MAS receptors was analyzed by Western Blotting. Results: Our AAA cohort consisted mostly males (76.6% AAA vs. 63.6% Control, p=0.68), smokers or ex-smokers (90.4% AAA vs. 36.3% Control, p=0.002) and most of them had hypertension(71.4% AAA vs. 36.3% Control, p=0.07). Analysis of protein expression in the AAA group showed that ACE and AT1 receptors were upregulated compared with Control (p=0.05; p=0.02, respectively,), while chymase expression was decreased in AAA compared to the control (p=0.03). Additionally, the AAA group showed downregulation in ACE-2 and AT2 compared with Control (respectively, p=0.01, p=0.03). There was no significant difference in the expression of renin and MAS receptors between groups (p=0.52, p=0.41, respectively,). Conclusions: Our data show that the ACE/AT1R axis was upregulated while ACE-2 and AT2 receptors were downregulated in the AAA aorta. Our results implicate local RAS in AAA development and warrant further studies.

Abstract Uniparental disomy (UPD) is the inheritance of both chromosomal homologs from one parent. Depending on the chromosome involved and the parental origin, UPD may result in phenotypic abnormalities due to aberrant methylation patterns or unmasking recessive conditions in isodisomic regions. UPD primarily originates from somatic rescue of a single meiotically‐derived aneuploidy, most commonly a trisomy. Double UPD is exceedingly rare and triple UPD has not been previously described. Here, we report two unrelated clinical cases with UPD of multiple chromosomes; an 8‐month‐old male with maternal isodisomy of chromosome 7 and paternal isodisomy of chromosome 9, and a 4‐week‐old female with mixed paternal UPD for chromosomes 4, 10, and 14. These cases also demonstrate that although extremely rare, the detection of AOH on two or more chromosomes may warrant additional clinical and laboratory investigation such as methylation and STR marker analysis, especially when involving chromosomes known to be associated with imprinting disorders.