Genotype-phenotype correlation: Inheritance and variant-type infer pathogenicity in IQSEC2 gene.

Pathogenic variants in the IQSEC2 gene including nonsense, frameshift, splice-alterations, deletions, and missense changes have been identified in individuals with X-linked mental retardation. Although highly variable, clinical features may include hypotonia, moderate to severe delayed psychomotor development, intellectual disability, speech deficits, refractory seizures, autistic features, and stereotypical movements. Females with de novo variants have been described with classical features. In contrast, the phenotype in carrier females identified through an affected male may range from asymptomatic to mild intellectual disability. We present male (N = 2) and female (N = 3) probands ascertained via diagnostic exome sequencing with distinct variant types in the IQSEC2 gene encompassing a spectrum of phenotypic severity with patient sex, variant type and inheritance hypothesized to drive disease penetrance and expressivity. All of these patients demonstrated epilepsy, global developmental delays, intellectual disability, and constipation. Our data support that de novo, truncating variants correlate with severe disease in both female and male patients harboring an IQSEC2 alteration. Missense variants in male and female patients may account for a milder disease overall, with more severe symptoms in males than females. We also present the first confirmed case of parental mosaicism, which has implications regarding counseling for recurrence risk. These data further delineate a genotype-phenotype correlation of IQSEC2 variation.

De novo loss-of-function variants in NSD2 (WHSC1) associate with a subset of Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) is a rare but recurrent microdeletion syndrome associated with hemizygosity of an interstitial segment of Chromosome 4 (4p16.3). Consistent with historical reports in which overlapping deletions defined a minimal critical region in WHS patients, recent reports from exome sequence analysis have provided further evidence that haploinsufficiency of a specific gene within this critical region, NSD2 (WHSC1), is causal for many features of the syndrome. In this report, we describe three unrelated patients with loss-of-function alterations in NSD2 who presented clinically with WHS features including intrauterine growth retardation and global developmental delay. Two of the three patients also had overlapping features of failure to thrive, short stature, constipation, and hypotonia. This series adds additional cases to expand the phenotypic spectrum of WHS and reports novel NSD2 variants.

Maternal obesity and gestational weight gain are modestly associated with umbilical cord DNA methylation.

INTRODUCTION: Maternal obesity (OB) and excessive gestational weight gain (GWG) are strong independent contributors that augment obesity risk in offspring. However, direct evidence of epigenetic changes associated with maternal habitus remains sparse. METHODS: We utilized Bisulfite Amplicon Sequencing (BSAS) to conduct targeted DNA methylation association analysis of maternal obesity and excessive GWG with DNA methylation of select metabolism-related and imprinted genes. Umbilical cord (UC) tissue from infants born to normal weight and overweight/obese women from the Glowing study were utilized (n = 78). RESULTS: In multivariable linear regression adjusted for relevant confounders, Institute on Medicine (IOM) GWG category and infant sex were significantly associated with UC IGFBP1 methylation, while gestation length was significantly associated with UC PRKAA1 methylation. In addition, infant fat mass (%) at 2 weeks of age was significantly associated with umbilical cord methylation of RAPTOR. While regression tree analysis confirmed findings from multivariable models demonstrating that maternal early pregnancy BMI and IOM GWG category are associated with fetal UC DNA methylation patterns for select metabolic and imprinted genes, in general, effect sizes were quite small and statistical significance was not maintained when accounting for multiple testing. DISCUSSION: Our findings suggest that maternal obesity and excessive GWG are weakly correlated with offspring DNA methylation patterns at birth.

Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing.

Cardiovascular and genitourinary anomalies in patients with duplications within the Williams syndrome critical region: phenotypic expansion and review of the literature.

Williams syndrome results from a microdeletion of approximately 1.5 Mb of chromosome 7q11.23. Several patients have been reported with the reciprocal microduplication in association with a variety of phenotypic features including cognitive impairment and typical facial features, though only a few have had birth defects. We report on three probands with duplications within 7q11.23 of variable sizes; two with cardiovascular involvement including aortic dilation and the other with unilateral renal and gonadal agenesis. We offer a comparison with previously reported cases of duplications of 7q11.23. In light of the present cases, we recommend undertaking echocardiographic and renal ultrasound evaluation of patients with documented 7q11.23 duplications. Further, this cytogenetic abnormality should be part of the differential diagnosis for patients with aortic dilation, as well as those with unilateral renal and gonadal agenesis.

Maternal FMR1 premutation allele expansion and contraction in fraternal twins.

Fragile X syndrome results from an expansion of the CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Expansion of a maternal premutation allele is the mechanism by which a full mutation allele arises; contraction of a maternal premutation allele is rare. Here we report on both an expansion and contraction of a maternal FMR1 premutation allele in fraternal twins. The propositus was the product of a 29-week gestation twin pregnancy and was referred for FMR1 testing due to developmental delay. A FMR1 full mutation with complete methylation was observed on Southern blot analysis. Evaluation of the maternal FMR1 gene by PCR revealed a normal and premutation allele with CGG repeat numbers of 30 and 93, respectively. Subsequent FMR1 testing on the twin sister of the propositus detected CGG repeat numbers of 30 and 54. The FMR1 CGG repeat number of the reproductive partner was 30. The FMR1 CGG repeat 30 allele in the twin sister was determined to be of paternal origin and the FMR1 allele with a CGG repeat number of 54 was of maternal origin. This observation is particularly interesting not only because of the concomitant donation of a FMR1 expanded and contracted premutation allele in a twin pregnancy but also because of the significant degree of contraction (39 repeats) of the maternal premutation allele.

A physiological role for connective tissue growth factor in early wound healing.

Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue growth factor (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and fibroblasts, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1-6) of tissue repair, prolonged administration (>15 days) of exogenous CTGF into PVA sponges resulted in fibroblast proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0-7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

Lessons from genetically altered mesenchymal stem cells (MSCs): candidates for improved MSC-directed myocardial repair.

The regenerative and reparative potential of mesenchymal stem cells (MSCs) make them attractive candidates for numerous cell-directed therapies. The variant degree of tissue repair by transplanted MSCs has been assessed in several published reports. There are many gaps in the knowledge of MSC biology and the underlying reasons for their disparate effectiveness in tissue repair. This review examines successful preclinical models of MSC-directed repair, particularly of myocardial repair, in an attempt to shed light into the events dictating MSC therapeutic efficacy. The reparative advantage of genetically altered MSCs will be described. This overview will elucidate possible molecular mechanisms that can influence MSC engraftment, differentiation, self-renewal, and ultimately increase wound repair.

Increased oxygen consumption and OXPHOS potential in superhealer mesenchymal stem cells.

BACKGROUND: Cell-based therapies show promise in repairing cardiac tissue and improving contractile performance following a myocardial infarction. Despite this, ischemia-induced death of transplanted cells remains a major hurdle to the efficacy of treatment. 'Superhealer' MRL/MpJ mesenchymal stem cells (MRL-MSCs) have been reported to exhibit increased engraftment resulting in reduced infarct size and enhanced contractile function. This study determines whether intrinsic differences in mitochondrial oxidative phosphorylation (OXPHOS) assist in explaining the enhanced cellular survival and engraftment of MRL-MSCs. FINDINGS: Compared to wild type MSCs (WT-MSCs), mitochondria from intact MRL-MSCs exhibited an increase in routine respiration and maximal electron transport capacity by 2.0- and 3.5-fold, respectively. When routine oxygen utilization is expressed as a portion of maximal cellular oxygen flux, the MRL-MSCs have a greater spare respiratory capcity. Additionally, glutamate/malate succinate-supported oxygen consumption in permeabilized cells was elevated approximately 1.25- and 1.4-fold in the MRL-MSCs, respectively. CONCLUSION: The results from intact and permeabilized MSCs indicate MRL-MSCs exhibit a greater reliance on and capacity for aerobic metabolism. The greater capacity for oxidative metabolism may provide a protective effect by increasing ATP synthesis per unit substrate and prevent glycolysis-mediated acidosis and subsequent cell death upon transplantation into the glucose-and oxygen-deprived environment of the infarcted heart.

Molecular mediators of mesenchymal stem cell biology.

Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate into multiple lineages making them an appropriate candidate for stem cell therapy. In spite of achieving considerable success in preclinical models, limited success has been achieved in clinical settings with MSCs. A major impediment that is faced is low survival of MSCs in injured tissues following implantation. In order to enhance the reparative properties of MSCs, it is vital to understand the molecular signals that regulate MSC survival and self-renewal. This review assimilates information that characterizes MSCs and mentions their utilization in myocardial infarction therapy. Additionally, our attempt herein is to gather pertinent published information regarding the role of canonical Wnt and BMP signaling in regulating the potential of MSCs to self-renew, proliferate, differentiate, and survive.

Pyrvinium, a potent small molecule Wnt inhibitor, promotes wound repair and post-MI cardiac remodeling.

Wnt signaling plays an important role in developmental and stem cell biology. To test the hypothesis that temporary inhibition of Wnt signaling will enhance granulation tissue and promote angiogenesis in tissue repair, we employed a recently characterized small molecule Wnt inhibitor. Pyrvinium is an FDA-approved drug that we identified as a Wnt inhibitor in a chemical screen for small molecules that stabilize ß-catenin and inhibit Axin degradation. Our subsequent characterization of pyrvinium has revealed that its critical cellular target in the Wnt pathway is Casein Kinase 1α. Daily administration of pyrvinium directly into polyvinyl alcohol (PVA) sponges implanted subcutaneously in mice generated better organized and vascularized granulation tissue; this compound also increased the proliferative index of the tissue within the sponges. To evaluate its effect in myocardial repair, we induced a myocardial infarction (MI) by coronary artery ligation and administered a single intramyocardial dose of pyrvinium. Mice were evaluated by echocardiography at 7 and 30 days post-MI and treatment; post mortem hearts were evaluated by histology at 30 days. Pyrvinium reduced adverse cardiac remodeling demonstrated by decreased left ventricular internal diameter in diastole (LVIDD) as compared to a control compound. Increased Ki-67+ cells were observed in peri-infarct and distal myocardium of pyrvinium-treated animals. These results need to be further followed-up to determine if therapeutic inhibition of canonical Wnt may avert adverse remodeling after ischemic injury and its impact on myocardial repair and regeneration.

sFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair.

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells.

Recent studies have identified a subpopulation of highly tumorigenic cells with stem/progenitor cell properties from human breast cancers, and it has been suggested that stem/progenitor cells, which remain after breast cancer therapy, may give rise to recurrent disease. We hypothesized that progenitor cells are resistant to radiation, a component of conventional breast cancer therapy, and that that resistance is mediated at least in part by Wnt signaling, which has been implicated in stem cell survival. To test this hypothesis, we investigated radioresistance by treating primary BALB/c mouse mammary epithelial cells with clinically relevant doses of radiation and found enrichment in normal progenitor cells (stem cell antigen 1-positive and side population progenitors). Radiation selectively enriched for progenitors in mammary epithelial cells isolated from transgenic mice with activated Wnt/beta-catenin signaling but not for background-matched controls, and irradiated stem cell antigen 1-positive cells had a selective increase in active beta-catenin and survivin expression compared with stem cell antigen 1-negative cells. In clonogenic assays, colony formation in the stem cell antigen 1-positive progenitors was unaffected by clinically relevant doses of radiation. Radiation also induced enrichment of side population progenitors in the human breast cancer cell line MCF-7. These data demonstrate that, compared with differentiated cells, progenitor cells have different cell survival properties that may facilitate the development of targeted antiprogenitor cell therapies.

Wnt/beta-catenin mediates radiation resistance of Sca1+ progenitors in an immortalized mammary gland cell line.

The COMMA-Dbeta-geo cell line has been shown to contain a permanent subpopulation of progenitor cells that are enriched in outgrowth potential. Using the COMMA-Dbeta-geo cell line as a model, we sought to study the radioresistance of mammary progenitor cells. Using the putative progenitor cell marker stem cell antigen 1 (Sca1), we were able to isolate a discrete subpopulation of Sca1(+) multipotent cells from the immortalized COMMA-Dbeta-geo murine mammary cell line. At a clinically relevant dose, the Sca1(+) cells were resistant to radiation (2 Gy). Sca1(+) cells contained fewer gamma-H2AX(+) DNA damage foci following irradiation, displayed higher levels of endogenous beta-catenin, and selectively upregulated survivin after radiation. Expression of active beta-catenin enhanced self-renewal preferentially in the Sca1(+) cells, whereas suppressing beta-catenin with a dominant negative, beta-engrailed, decreased self-renewal of the Sca1(+) cells. Understanding the radioresistance of progenitor cells may be an important factor in improving the treatment of cancer. The COMMA-Dbeta-geo cell line may provide a useful model to study the signaling pathways that control mammary progenitor cell regulation.