Generation of Urine-Derived Induced Pluripotent Stem Cells and Cerebral Organoids for Modeling Down Syndrome.

Down syndrome (DS, or trisomy 21, T21), is the most common genetic cause of intellectual disability. Alterations in the complex process of cerebral cortex development contribute to the neurological deficits in DS, although the underlying molecular and cellular mechanisms are not completely understood. Human cerebral organoids (COs) derived from three-dimensional (3D) cultures of induced pluripotent stem cells (iPSCs) provide a new avenue for gaining a better understanding of DS neuropathology. In this study, we aimed to generate iPSCs from individuals with DS (T21-iPSCs) and euploid controls using urine-derived cells, which can be easily and noninvasively obtained from most individuals, and examine their ability to differentiate into neurons and astrocytes grown in monolayer cultures, as well as into 3D COs. We employed nonintegrating episomal vectors to generate urine-derived iPSC lines, and a simple-to-use system to produce COs with forebrain identity. We observed that both T21 and control urine-derived iPSC lines successfully differentiate into neurons and astrocytes in monolayer, as well as into COs that recapitulate early features of human cortical development, including organization of neural progenitor zones, programmed differentiation of excitatory and inhibitory neurons, and upper-and deep-layer cortical neurons as well as astrocytes. Our findings demonstrate for the first time the suitability of using urine-derived iPSC lines to produce COs for modeling DS.

The Urinary Peptidome as a Noninvasive Biomarker Development Strategy for Prenatal Screening of Down's Syndrome.

Prenatal screening for Down's syndrome based on maternal age, ultrasound measures, and maternal serum biomarkers is recommended worldwide, but the false-positive rate and poor diagnostic performance of these screening tests remain problematic. Genetic analysis of cell-free DNA in maternal blood has been developed as a new prenatal screening for Down's syndrome, but it has a number of limitations, including turnaround time and cost. Prenatal screening diagnostic innovation calls for new tests that are noninvasive, accurate, and affordable. We report original observations on potential peptide biomarkers in maternal urine for screening of fetal Down's syndrome. The peptidome of urine samples from 23 pregnant women carrying Down's syndrome fetuses and 30 pregnant women carrying fetuses with normal karyotype was fractionated by weak cation exchange magnetic beads and analyzed by MALDI-TOF mass spectrometry. Levels of six peptides (m/z 1022.1, 1032.1, 1099.5, 1155.9, 1306.6, and 2365.6) were significantly altered between the case and control groups after controlling for maternal and gestational age. A classification model was constructed based on these candidate peptides that could differentiate fetuses with Down's syndrome from controls with a sensitivity of 95.7%, a specificity of 70.0%, and an area under receiver operating characteristic curves of 0.909 (95% confidence interval, 0.835-0.984). Peptide peaks at m/z 1099.5 and 1155.9 were identified as the partial sequences of alpha-1-antitrypsin and heat shock protein beta-1, respectively. These new findings support the new idea that maternal urinary peptidome offers prospects for noninvasive biomarker discovery and development for the prenatal screening of fetal Down's syndrome.

Urinary biomarkers and obstructive sleep apnea in patients with Down syndrome.

STUDY OBJECTIVES: The study aimed to compare urinary biomarkers in patients with Down syndrome (DS) with and without obstructive sleep apnea (OSA) to those of age- and sex-matched neurotypically developing healthy controls (HC). We further investigated whether we could predict OSA in patients with DS using these biomarkers. METHODS: Urine samples were collected from 58 patients with DS the night before or the morning after their scheduled overnight polysomnogram or both, of whom 47 could be age- and sex-matched to a sample of 43 HC. Concentrations of 12 neurotransmitters were determined by enzyme-linked immunosorbent assay. Log-transformed creatinine-corrected assay levels were normalized. Normalized z-scores were compared between patients with DS vs. HC, between patients with DS with vs. without OSA, and to derive composite models to predict OSA. RESULTS: Most night-sampled urinary biomarkers were elevated among patients with DS relative to matched HC. No urinary biomarker levels differed between patients with DS with vs. without OSA. A combination of four urinary biomarkers predicted AHI > 1 with a positive predictive value of 90% and a negative predictive value of 68%. CONCLUSIONS: Having DS, even in the absence of concurrent OSA, is associated with a different urinary biomarker profile when compared to that of HC. Therefore, while urinary biomarkers may be predictive of OSA in the general pediatric population, a different approach is needed in interpreting urinary biomarker assays in patients with DS. Certain biomarkers also seem promising to be predictive of OSA in patients with DS. No clinical trial was indicated in the undertaking of this work.

Generation of Integration-Free Induced Pluripotent Stem Cells from Urine-Derived Cells Isolated from Individuals with Down Syndrome.

Down syndrome (DS) is a genetic disorder caused by trisomy 21 (T21). Over the past two decades, the use of mouse models has led to significant advances in the understanding of mechanisms underlying various phenotypic features and comorbidities secondary to T21 and even informed the design of clinical trials aimed at enhancing the cognitive abilities of persons with DS. In spite of its success, this approach has been plagued by all the typical limitations of rodent modeling of human disorders and diseases. Recently, several laboratories have succeeded in producing T21 human induced pluripotent stem cells (T21-iPSCs) from individuals with DS, which is emerging as a promising complementary tool for the study of DS. Here, we describe the method by which we generated 10 T21-iPSC lines from epithelial cells in urine samples, presumably from kidney epithelial origin, using nonintegrating episomal vectors. We also show that these iPSCs maintain chromosomal stability for well over 20 passages and are more sensitive to proteotoxic stress than euploid iPSCs. Furthermore, these iPSC lines can be differentiated into glutamatergic neurons and cardiomyocytes. By culturing urine-derived cells and maximizing the efficiency of episomal vector transfection, we have been able to generate iPSCs noninvasively and effectively from participants with DS in an ongoing clinical trial, and thus address most shortcomings of previously generated T21-iPSC lines. These techniques should extend the application of iPSCs in modeling DS and other neurodevelopmental and neurodegenerative disorders, and may lead to future human cell-based platforms for high-throughput drug screening. Stem Cells Translational Medicine 2017;6:1465-1476.

Cognitive ability in Down syndrome and its relationship to urinary neopterin, a marker of activated cellular immunity.

BACKGROUND: Neopterin is an unconjugated pteridine that is secreted in large quantities by activated macrophages and can be used as a clinical marker of activated cellular immunity and oxidative stress. We aimed to investigate whether urinary neopterin levels are associated with cognitive function in people with Down syndrome (DS). METHODS: Out of 32 adults with DS who originally participated in a longitudinal study, 25 were followed up at 4 years. Informants rated their adaptive behavior (ABAS) and the adults with DS attempted assessments of language skills and memory at both baseline and follow-up time points (Modified Memory Object Task, MOMT), and receptive vocabulary (British Picture Vocabulary Scale, BPVS). RESULTS: Neopterin/creatinine levels were negatively correlated with change in the MOMT total score (Spearman's Rho=-0.517, p=0.020) and change in the MOMT delayed recall score (Spearman's Rho=-0.577, p=0.008) over time, i.e. higher neopterin/creatinine level was associated with worse performance on a test of cognitive ability over time. CONCLUSION: Urine neopterin may have potential as a biomarker for memory decline in Down syndrome, and could potentially also help to track progression of mild cognitive impairment (MCI) to Alzheimer's disease in other high risk populations.

Shotgun metabolomic profiles in maternal urine identify potential mass spectral markers of abnormal fetal biochemistry - dihydrouracil and progesterone in the metabolism of Down syndrome.

In Down syndrome (DS) in particular, the precise cellular mechanisms linking genotype to phenotype is not straightforward despite a clear mapping of the genetic cause. Metabolomic profiling might be more revealing in understanding molecular-cellular mechanisms of inborn errors of metabolism/syndromes than genomics alone and also result in new prenatal screening approaches. The urinary metabolome of 122 maternal urine from women with and without an aneuploid pregnancy (predominantly Down syndrome) were compared by both zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and reversed-phase liquid chromatography (RPLC) coupled to hybrid ion trap time of flight mass spectral analysis. ZIC-HILIC mass spectrometry resolved 10-fold more unique molecular ions than RPLC mass spectrometry, of which molecules corresponding to ions of m/z 114.07 and m/z 314.20 showed maternal urinary level changes that significantly coincided with the presence of a DS fetus. The ion of m/z 314.20 was identified as progesterone and m/z 114.07 as dihydrouracil. A metabolomics profiling-based maternal urinary screening test modelled from this separation data would detect approximately 87 and 60.87% (using HILIC-MS and RPLC-MS, respectively) of all DS pregnancies between 9 and 23 weeks of gestation with no false positives.

Memory decline in Down syndrome and its relationship to iPF2alpha, a urinary marker of oxidative stress.

BACKGROUND: Lipid peroxidation may be a marker of free-radical-mediated injury associated with Alzheimer's disease (AD). We aimed to investigate whether changes in lipid peroxidation is associated with cognitive decline in individuals with Down syndrome over a 4-year period. METHODS: Thirty-two adults with DS participated in a longitudinal study with urinary isoprostane 8,12-iso-iPF2alpha (iPF2alpha) assays at baseline and four years follow-up. Informants rated their functional ability and memory function and the adults with DS attempted assessments of language skills and memory. Twenty-six individuals completed assessments of memory (Modified Memory Object Task, MOMT), adaptive behavior (ABAS), and receptive vocabulary (British Picture vocabulary, BPVS) at both time-points. RESULTS: Overall change in iPF2alpha level was negatively correlated with change in the MOMT score (Spearman's Rho =  -0.576, p = 0.006), i.e., increased lipid peroxidation was correlated with worse memory functioning over time. An increase of ≥ 0.02 ng/mg creatinine iPF2α had good sensitivity (85.7%), positive predictive value (75%,), specificity (85.7%) and negative predictive value (92.3%) for memory decline. CONCLUSION: Change in iPF2alpha over time may have potential as a biomarker for memory decline in Down syndrome and potentially also help to track progression of MCI to AD in the general population.

Second trimester maternal urine for the diagnosis of trisomy 21 and prediction of poor pregnancy outcomes.

Given the recognized lack of prenatal clinical methods for the early diagnosis of preterm delivery, intrauterine growth restriction, preeclampsia and gestational diabetes mellitus, and the continuing need for optimized diagnosis methods for specific chromosomal disorders (e.g., trisomy 21) and fetal malformations, this work sought specific metabolic signatures of these conditions in second trimester maternal urine, using (1)H Nuclear Magnetic Resonance ((1)H NMR) metabolomics. Several variable importance to the projection (VIP)- and b-coefficient-based variable selection methods were tested, both individually and through their intersection, and the resulting data sets were analyzed by partial least-squares discriminant analysis (PLS-DA) and submitted to Monte Carlo cross validation (MCCV) and permutation tests to evaluate model predictive power. The NMR data subsets produced significantly improved PLS-DA models for all conditions except for pre-premature rupture of membranes. Specific urinary metabolic signatures were unveiled for central nervous system malformations, trisomy 21, preterm delivery, gestational diabetes, intrauterine growth restriction and preeclampsia, and biochemical interpretations were proposed. This work demonstrated, for the first time, the value of maternal urine profiling as a complementary means of prenatal diagnostics and early prediction of several poor pregnancy outcomes.

Predictors, including blood, urine, anthropometry, and nutritional indices, of all-cause mortality among institutionalized individuals with intellectual disability.

As the life expectancy of people with intellectual disability (ID) increases, it is becoming necessary to understand factors affecting survival. However, predictors that are typically assessed among healthy people have not been examined. Predictors of all-cause mortality, including blood, urine, anthropometry, and nutritional indices, were examined among institutionalized people with ID. This retrospective cohort study involved 316 participants (191 males, 125 females; mean age, 36.5 ± 10.5 years) at a public facility for people with ID in Ibaraki Prefecture, Japan. During the follow-up from the examination day in 1984-1992 through December 31, 2007 (mean follow-up, 18.6 years), 44 deaths occurred. Mean age at death was 47.1 ± 10.0 years (range, 22.3-65.3 years). Early deaths within three years (n = 4) were treated as censored cases. Cox proportional hazard regression models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause mortality. Sex- and age-adjusted analysis (p<0.15) revealed positive associations with mortality for high serum cholesterol, high thymol turbidity test (TTT), and glucosuria and negative associations with mortality for high serum albumin, high uric acid, high potassium, high calcium, and high systolic blood pressure. Multivariate analysis revealed that male sex (HR, 4.11; 95% CI, 1.59-10.59), high serum cholesterol (1.01; 1.00-1.02), high serum TTT (1.21; 1.03-1.41), and epilepsy significantly increased the mortality risk. The results indicate that the predictors of life expectancy for people with ID included both factors that are shared with healthy people (male sex, high serum cholesterol) and factors specific to people with disabilities (high serum TTT and epilepsy).

Systemic oxidative stress, as measured by urinary allantoin and F(2)-isoprostanes, is not increased in Down syndrome.

PURPOSE: Oxidative stress has been implicated in Down syndrome (DS) pathology. This study compares DS individuals and controls on their urinary levels of allantoin and 2,3-dinor-iPF2α-III; these biomarkers have been previously validated in a clinical model of oxidative stress. METHODS: Urine samples were collected from 48 individuals with DS and 130 controls. Biomarkers were assayed by ultraperformance liquid chromatography-tandem mass spectrometry, normalized by urinary creatinine concentration. RESULTS: After adjusting for age and gender, mean allantoin levels were lower among DS individuals versus controls (P = .04). The adjusted mean levels of 2,3-dinor-iPF2α-III were similar in DS individuals and controls (P = .7). CONCLUSIONS: Our results do not support the hypothesis that DS individuals have chronic systemic oxidative stress.

Evaluation of urinary biomarkers of oxidative/nitrosative stress in children with Down syndrome.

AIMS: It has been suggested that oxidative stress plays a key role in the pathogenesis of Down syndrome (DS). However, urinary biomarkers of oxidative stress have been little studied in this condition. Thus, we aimed to assess a set of urinary oxidative/nitrosative stress biomarkers in children with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2'-deoxyguanosine (8-OHdG), isoprostane 15-F(2t)-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H(2)O(2)) and nitrite/nitrate (NOx). MAIN METHODS: Fluorimetric and spectrophotometric assays were performed in children with DS (n=26), some of them taking levothyroxine for hypothyroidism (n=7), and their non-Down siblings (n=19). KEY FINDINGS: We found that only levels of diTyr were increased in DS, although no differences were obtained when hypothyroid DS children were excluded. Levels of 8-OHdG, 15-F(2t)-IsoP, TBARS, AGEs, H(2)O(2) and NOx did not differ neither between DS and controls nor between hypothyroid DS children and DS without hypothyroidism diagnosed. However, diTyr is increased in hypothyroid DS children compared with controls. Negative correlations with age were obtained for 8-OHdG, diTyr and NOx in DS and controls and for 8-OHdG, 15-F(2t)-IsoP, TBARS and AGEs in DS. SIGNIFICANCE: Increased oxidative stress in children with DS cannot be explained by the urinary levels of 8-OHdG, 15-F(2t)-IsoP, TBARS, AGEs, diTyr, H(2)O(2) and NOx, at least with the assays used. Nonetheless, urinary diTyr could be used as oxidative/nitrosative stress biomarker in hypothyroid DS children. The present work presents evidence of a probable renal impairment in children with DS receiving levothyroxine for hypothyroidism.

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome.

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2'-deoxyguanosine (8-OHdG), isoprostane 15-F(2t)-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H(2)O(2)) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n=78), some of them taking levothyroxine for hypothyroidism (n=24), and in their healthy age-matched controls (n=65). We found that levels of AGEs, diTyr, H(2)O(2) and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F(2t)-IsoP and NOx. We also found lower levels of Cr from 15 to 19years, higher levels of TBARS and AGEs from 20 to 40years and higher levels of diTyr from 15 to 40years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H(2)O(2) and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F(2t)-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.

Melatonin and elimination of kynurenines in children with Down's syndrome.

BACKGROUND: Heightened activity of superoxide dimutase is an effect derived from the gene dose in the trisomy of Down's syndrome (DS), and has been related to the increased production of hydrogen peroxide and with greater lipid peroxidation. Many of the degenerative changes observed in patients with DS have been associated with the pathological effects of free radicals, and for this reason it is of interest to determine the levels present in these patients of powerful antioxidant molecules such as melatonin, and of metabolites with important neuroprotector and neurotoxic consequences such as those derived from the kynurenine pathway. PATIENTS AND METHODS: A study was made of 15 children with DS, together with a control group of 15 non-DS children, matched for age and sex, examined at the Hospital Costa del Sol, Marbella, Spain. Serum melatonin and serotonin were analyzed by RIA; urinary tryptophan metabolites (kynurenine pathway) were determined during periods of light and darkness (09.00-21.00 h and 21.00-9.00 h) by thin-layer chromatography. RESULTS: The mean values of serotonin and melatonin were found to be lower in the patients with DS, although the level of nocturnal secretion of melatonin was higher. Urinary excretion of kynurenine was lower in the patients with DS, although greater quantities of kynurenic acid and anthranilic acid were excreted. CONCLUSIONS: Patients with DS present levels of plasma melatonin and urinary kynurenine that are lower than the corresponding levels in the control population, together with higher values of kynurenic acid and anthranilic acid. These circumstances constitute an added risk to these patients of damage by free radicals.

Urinary uric acid and antioxidant capacity in children and adults with Down syndrome.

OBJECTIVES: To evaluate the urinary levels of uric acid (UA) and total antioxidant capacity (TAC) with and without UA relative contribution (TAC(-UA)) in children and adults with Down syndrome (DS) and to prove the clinical use of TAC. DESIGN AND METHODS: Urine samples were obtained from 32 individuals with DS and 29 controls. Two age groups were established (children and adults). Spectrophotometric methods were used for biochemical determinations. RESULTS: Children with DS had significantly higher UA/Cr and TAC/Cr levels than controls, whereas levels of TAC(-UA)/Cr were lower in adults with DS than in controls (P<0.05 for all). In DS, levels of UA/Cr, TAC/Cr and TAC(-UA)/Cr were higher in children than in adults (P<0.05 for all). Positive correlations between UA/Cr and TAC/Cr were found for all groups studied. Negative correlations with age were found for UA/Cr and TAC/Cr in children of both groups. CONCLUSIONS: Our results proved that TAC is decreased in adults with DS. Besides, TAC(-UA) seems to provide more reliable information about the antioxidant status, at least in DS.

SURUSS in perspective.

BACKGROUND: Until the publication of the Serum Urine and Ultrasound Screening Study (SURUSS) report, it was difficult to compare the different antenatal screening tests for Down's Syndrome because of variations in study designs. We here present the main results from SURUSS, updated to take account of recent information on nuchal translucency in Down's Syndrome pregnancies, and discuss their implications. METHODS: SURUSS was a prospective study of 47,053 singleton pregnancies (including 101 pregnancies with Down's Syndrome) conducted in 25 maternity units. Nuchal translucency measurements were taken. Serum and urine samples collected between 9 and 13 weeks, and again between 14 and 20 weeks of pregnancy were stored. Samples from each affected pregnancy and five matched controls were tested for currently used or suggested biochemical Down's Syndrome screening markers. Pregnancies were followed up to determine the presence or absence of Down's Syndrome. For an 85% Down's Syndrome detection rate, the false-positive rate for the Integrated test (nuchal translucency and pregnancy associated plasma protein-A [PAPP-A] at 11 completed weeks of pregnancy, and alpha-fetoprotein, unconjugated oestriol [uE3], free beta or total human chorionic gonadotrophin (hCG) and inhibin-A in the early second trimester) was 0.9%, the Serum integrated test (without nuchal translucency) 2.7%, the Combined test (nuchal translucency with free beta-hCG and PAPP-A at 11 weeks) 4.3%, the Quadruple test (alpha-fetoprotein, uE3, free beta or total hCG and inhibin-A) 6.2%, and nuchal translucency at 11 weeks, 15.2%. All tests included maternal age. Using the Integrated test at an 85% detection rate, there would be six diagnostic procedure-related unaffected fetal losses following amniocentesis per 100,000 women screened compared with 35 using the Combined test or 45 with the Quadruple test. CONCLUSIONS: The Integrated test offers the most effective and safe method of screening for women who attend in the first trimester. The next best test is the Serum integrated test. The Quadruple test is the best test for women who first attend in the second trimester. There is no justification for retaining the Double (alpha-fetoprotein and hCG) or Triple (alpha-fetoprotein, uE3, and hCG) tests, or nuchal translucency alone (with or without maternal age) in antenatal screening for Down's Syndrome.

Prenatal screening tests facilitate risk assessment.

Medical technology continues to improve services and testing to provide accurate and timely information for physicians and patients. New screening protocols in which information obtained in the first and second trimesters is integrated to improve detection and lower false positive rates have potential for safer and more effective prenatal care. Families still have difficult choices to make when confronted with the diagnosis of a birth defect. Inherent advantages of early detection are reassurance for low-risk women sooner, and for women with increased risk, more time to consider all options. Without doubt, improvements in risk assessment, diagnosis, and prevention will continue. The advantage offered by laboratory procedures is that the preliminary diagnosis can be made by simple, noninvasive screening tests.