Case of an unreported genetic variant of salt losing 3-ß-hydroxysteroid dehydrogenase deficiency.

Salt losing 3-ß-hydroxysteroid dehydrogenase deficiency (HSD3B2) is a rare form of congenital adrenal hyperplasia, seen in <0.5% of cases. We present a 7-year-old male diagnosed with HSD3B2 deficiency, not identified by state newborn screen, due to a novel variant identified in the HSD3B2 gene (c.694C > G; p.His232Asp). This patient was referred to pediatric endocrinology and pediatric biochemical genetics following a fourth hospitalization for emesis and electrolyte derangements including hyponatremia, hyperkalemia, ketoacidosis and hypoglycemia. Endocrinology evaluation yielded elevated 17-hydroxyprogesterone (17-OHP), 17-hydroxypregnenolone (17-OHPreg), dehydroepiandrosterone and adrenocorticotropic hormone (ACTH). ACTH stimulation test indicated flat response. Sequencing of the HSD3B2 revealed a pathogenic variant inherited in trans with the novel c.694C > G (p.His232Asp) variant. The patient was started on daily glucocorticoid and mineralocorticoid replacement and has since had no further adrenal crises.

Rapid whole-genome sequencing identifies a homozygous novel variant, His540Arg, in HSD17B4 resulting in D-bifunctional protein deficiency disorder diagnosis.

Rapid whole-genome sequencing (rWGS) allows for a diagnosis to be made quickly and impact medical management, particularly in critically ill children. Variants identified by this approach are often not identified using other testing methodologies, such as carrier screening or gene sequencing panels, targeted panels, or chromosomal microarrays. However, rWGS can identify variants of uncertain significance (VUSs), which challenges clinicians in the rapid return of information to families. Here we present a case of the metabolic condition D-bifunctional protein deficiency in a neonate with epilepsy and hypotonia born to consanguineous parents. Sequencing revealed a homozygous VUS in HSD17B4, c.1619A > G (p.His540Arg). Preliminary results were delivered within 3 d of sample receipt. Previous parental carrier screening included the HSD17B4 gene but was reported as negative. The molecular finding directed the clinical team to assess phenotypic overlap and investigate next steps in terms of confirmation of the findings and potential medical management of the patient. Clinical metabolic testing of fatty acids confirmed the diagnosis. Computational analysis of HSD17B4 His540Arg showed the change to likely impact dimerization based on structural insights, with the histidine conserved and selected throughout all 223 species assessed for this amino acid. This variant clusters around several pathogenic and likely pathogenic variants in HSD17B4 This case demonstrates the utility of rWGS, the potential for receiving uncertain results, and the downstream implications for confirmation or rejection of a molecular diagnosis by the clinical team.

Recurrent deletions and duplications of chromosome 2q11.2 and 2q13 are associated with variable outcomes.

Copy number variation (CNV) in the long arm of chromosome 2 has been implicated in developmental delay (DD), intellectual disability (ID), autism spectrum disorder (ASD), congenital anomalies, and psychiatric disorders. Here we describe 14 new subjects with recurrent deletions and duplications of chromosome 2q11.2, 2q13, and 2q11.2-2q13. Though diverse phenotypes are associated with these CNVs, some common features have emerged. Subjects with 2q11.2 deletions often exhibit DD, speech delay, and attention deficit hyperactivity disorder (ADHD), whereas those with 2q11.2 duplications have DD, gastroesophageal reflux, and short stature. Congenital heart defects (CHDs), hypotonia, dysmorphic features, and abnormal head size are common in those with 2q13 deletions. In the 2q13 duplication cohort, we report dysmorphic features, DD, and abnormal head size. Two individuals with large duplications spanning 2q11.2-2q13 have dysmorphic features, hypotonia, and DD. This compilation of clinical features associated with 2q CNVs provides information that will be useful for healthcare providers and for families of affected children. However, the reduced penetrance and variable expressivity associated with these recurrent CNVs makes genetic counseling and prediction of outcomes challenging. © 2015 Wiley Periodicals, Inc.

Direct-to-consumer genomic testing offers little clinical utility but appears to cause minimal harm.

Direct-to-consumer genomic testing is available to anyone willing to pay for it. We investigated the reliability and reproducibility of such testing by sending DNA samples to 2 popular companies and by reviewing current literature on this topic. The concerns that were initially raised about direct-to- consumer genomic testing still seem valid.

EPC mobilization after erythropoietin treatment in acute ST-elevation myocardial infarction: the REVEAL EPC substudy.

Erythropoietin (EPO) was hypothesized to mitigate reperfusion injury, in part via mobilization of endothelial progenitor cells (EPCs). The REVEAL trial found no reduction in infarct size with a single dose of EPO (60,000 U) in patients with ST-segment elevation myocardial infarction. In a substudy, we aimed to determine the feasibility of cryopreserving and centrally analyzing EPC levels to assess the relationship between EPC numbers, EPO administration, and infarct size. As a prespecified substudy, mononuclear cells were locally cryopreserved before as well as 24 and 48-72 h after primary percutaneous coronary intervention. EPC samples were collected in 163 of 222 enrolled patients. At least one sample was obtained from 125 patients, and all three time points were available in 83 patients. There were no significant differences in the absolute EPC numbers over time or between EPO- and placebo-treated patients; however, there was a trend toward a greater increase in EPC levels from 24 to 48-72 h postintervention in patients receiving ≥30,000 U of EPO (P = 0.099 for CD133(+) cells, 0.049 for CD34(+) cells, 0.099 for ALDH(br) cells). EPC numbers at baseline were inversely related to infarct size (P = 0.03 for CD133(+) cells, 0.006 for CD34(+) cells). Local whole cell cryopreservation and central EPC analysis in the context of a multicenter randomized trial is feasible but challenging. High-dose (≥30,000 U) EPO may mobilize EPCs at 48-72 h, and baseline EPC levels may be inversely associated with infarct size.

The effect of aspirin on endothelial progenitor cell biology: preliminary investigation of novel properties.

UNLABELLED: Atherosclerosis develops in an environment of endothelial injury and inflammation. Circulating endothelial progenitor cells (EPCs) are required for vascular repair and restoration of normal endothelial function. We tested the hypothesis that the nonselective cyclooxygenase (COX) inhibitor aspirin (ASA) exerts an effect on circulating EPCs. METHODS: As part of a larger study evaluating the effect of aspirin dose in primary and secondary prevention, subjects (n=32) were assigned randomly to either 81 mg or 325 mg aspirin daily for two months, and circulating mononuclear cells were enumerated at the beginning of the study and after 2 months using fluorescent antibodies against CD34 and CD133 as well as based on aldehyde dehydrogenase (ALDH) activity. Brachial artery endothelial function via flow-mediated dilation (BAFMD) and light transmittance platelet aggregometry in response to physiologic agonists was also determined. RESULTS: Subjects taking aspirin at the time of study entry had a lower numbers of CD133+/34+ cells compared to those not previously exposed (0.01% vs. 0.05% of MNCs, P<0.03). After 2 months, subjects randomized to 81 vs. 325 mg of ASA had no significant differences in the median numbers of EPCs, although mean numbers trended lower in the high dose group. Patients on chronic ASA therapy continued to have lower numbers of EPCs. Similar effects were observed in CD34 and CD 133 single-positive cells, as well as ALDH(br) cells. BAFMD did not differ nor change significantly over time between aspirin dose groups. All patients had decreased ex vivo platelet aggregation in response to arachidonic acid and ADP stimulation. CONCLUSIONS: Our preliminary studies suggest that aspirin exerts a time-dependent effect on circulating EPCs. Short-term exposure to differing doses of ASA had indeterminate effects on EPCs levels, suggesting that time of ASA exposure may play a more important role than dose. Determining the responsible mechanism(s) and the overall clinical relevance of these findings will require further investigation.

Aging is not associated with bone marrow-resident progenitor cell depletion.

Changes in progenitor cell biology remain at the forefront of many theories of biologic aging, but there are limited studies evaluating this in humans. Aging has been associated with a progressive depletion of circulating progenitor cells, but age-related bone marrow-resident progenitor cell depletion has not been systematically determined in humans. Patients undergoing total hip replacement were consented, and bone marrow and peripheral progenitor cells were enumerated based on aldehyde dehydrogenase activity and CD34 and CD133 expression. Circulating progenitors demonstrated an age-dependent decline. In contrast, marrow-resident progenitor cell content demonstrated no age association with any progenitor cell subtype. In humans, aging is associated with depletion of circulating, but not marrow-resident, progenitors. This finding has impact on the mechanism(s) responsible for age-related changes in circulating stem cells and important implications for the use of autologous marrow for the treatment of age-related diseases.

Aldehyde dehydrogenase activity allows reliable EPC enumeration in stored peripheral blood samples.

BACKGROUND: Interest in the biology of endogenous progenitor cells (EPCs) continues to grow as evidence of their role in vascular repair mounts. EPC enumeration requires specialized laboratory techniques and is performed immediately after sample acquisition, limiting the clinical contexts in which EPC enumeration can be performed and the ability to increase sample sizes through multi-center participation. METHODS: We compared the numbers of EPCs enumerated in samples processed immediately after acquisition (n = 36) with EPCs enumerated in specimens stored for 24 hours or after cryopreservation of mononuclear cells (MNC) using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH(br) cells). RESULTS: EPCs assessed in fresh samples correlated with EPCs enumerated after whole blood storage (r = 0.699 for CD133(+)CD34(+) cells, r = 0.880 for ALDH(br) cells, P < 0.005 and P < 0.0001, respectively) or mononuclear cryopreservation (r = 0.590 for CD133(+)CD34(+) cells, r = 0.894 for ALDH(br) cells, P < 0.0001 for each); however, correlation based on assessment of ALDH(br) cells was higher (P < 0.0003 for comparison of correlation coefficients). Initial results from a multi-site clinical trial suggest that EPC enumeration after mononuclear cell cryopreservation is feasible. CONCLUSION: EPC analysis based on ALDH activity is reproducible, even after extended whole blood storage or MNC cryopreservation.