Valorisation of the invasive alga Rugulopteryx okamurae through the production of monomeric sugars.

Rugulopteryx okamurae is an invasive brown alga causing severe environmental and economic problems on the western Mediterranean coasts. Thus, in addition to the difficulties caused to the fishing and tourism sectors, there is a need to manage its accumulation on the beaches. This work aims to valorise this waste by using it as raw material for producing monosaccharides through a two-stage sequential process. These sugars could be used for different fermentative processes to obtain high-value-added bioproducts. In this work, biological pretreatment of the previously conditioned seaweed with the fungus Aspergillus awamori in solid-state fermentation (SSF), followed by enzymatic hydrolysis with a commercial enzyme cocktail, was performed. The effect of the extension of the biological pretreatment (2, 5, 8 and 12 days) on the subsequent release of total reducing sugars (TRS) in the enzymatic hydrolysis stage was studied. To analyse this effect, experimental data of TRS produced along the hydrolysis were fitted to simple first-order kinetics. Also, the secretion of cellulase and alginate lyase by the fungus, along with the biological pretreatment, was determined. The results suggest that 5 days of biological pretreatment of the macroalgae with A. awamori followed by enzymatic saccharification for 24 h with Cellic CTec2® (112 FP units/g of dry biomass) are the best conditions tested, allowing the production of around 240 g of TRS per kg of dried biomass. The main sugars obtained were glucose (95.8 %) and mannitol (1.5 %), followed by galactose (1 %), arabinose (0.9 %) and fucose (0.5 %). KEY POINTS: • Five-day SSF by A. awamori was the best condition to pretreat R. okamurae. • Five-day SSF was optimal for alginate lyase production (1.63 ±0.011 IU/g biomass). • A maximum yield of 239 mg TRS/g biomass was obtained (with 95.8 % glucose).

Clinical exome sequencing vs. usual care for hereditary colorectal cancer diagnosis: A pilot comparative effectiveness study.

BACKGROUND: Clinical exome sequencing (CES) provides the advantage of assessing genetic variation across the human exome compared to a traditional stepwise diagnostic approach or multi-gene panels. Comparative effectiveness research methods offer an approach to better understand the patient-centered and economic outcomes of CES. PURPOSE: To evaluate CES compared to usual care (UC) in the diagnostic work-up of inherited colorectal cancer/polyposis (CRCP) in a randomized controlled trial (RCT). METHODS: The primary outcome was clinical sensitivity for the diagnosis of inherited CRCP; secondary outcomes included psychosocial outcomes, family communication, and healthcare resource utilization. Participants were surveyed 2 and 4 weeks after results return and at 3-month intervals up to 1 year. RESULTS: Evolving outcome measures and standard of care presented critical challenges. The majority of participants in the UC arm received multi-gene panels [94.73%]. Rates of genetic findings supporting the diagnosis of hereditary CRCP were 7.5% [7/93] vs. 5.4% [5/93] in the CES and UC arms, respectively (P = 0.28). Differences in privacy concerns after receiving CRCP results were identified (0.88 in UC vs 0.38 in CES, P = 0.05); however, healthcare resource utilization, family communication and psychosocial outcomes were similar between the two arms. More participants with positive results (17.7%) intended to change their life insurance 1  month after the first return visit compared to participants returned a variant of uncertain significance (9.1%) or negative result (4.8%) (P = 0.09). CONCLUSION: Our results suggest that CES provides similar clinical benefits to multi-gene panels in the diagnosis of hereditary CRCP.

Next Generation Sequencing in the Clinic: a Patterns of Care Study in a Retrospective Cohort of Subjects Referred to a Genetic Medicine Clinic for Suspected Lynch Syndrome.

Next generation sequencing (NGS) gene panels are increasingly used in medical genetics clinics for the evaluation of common inherited cancer syndromes, but the clinical efficacy of these tests, and the factors driving clinical providers to order them are unclear. We conducted a patterns-of-care study to compare patients evaluated with NGS gene panels with a reference group. We abstracted demographic, socioeconomic, and clinical information in a retrospective cohort of patients referred to a large medical genetics clinic for evaluation of inherited colorectal cancer and polyposis syndromes. Patients tested with NGS gene panels were more likely to be insured compared to the reference group (85.3 % vs. 69.2 %, p = 0.0068),less likely to have prior tumor tissue testing (29.4 % vs. 54.3 %, p = 0.0004), and less likely to have an abnormal tumor tissue test result (46.7 % vs. 74.5 %, p = 0.01). No significant differences were found between groups in age, gender, race, employment status, personal history of colorectal cancer, or proportion of patients fulfilling Lynch syndrome clinical criteria. Patients with NGS testing were less likely to have a pathogenic/likely pathogenic variant detected (13.7 % vs. 31.9 %, p = 0.002). Patients referred for NGS testing to evaluate inherited colorectal cancer/polyposis risk appear to undergo tumor tissue testing less frequently than non-NGS testing patients. Further studies are needed to assess the most effective and cost-effective approach to genomic diagnosis in this patient population.

Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network.

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data.

Thermophilic anaerobic co-digestion of organic fraction of municipal solid waste (OFMSW) with food waste (FW): Enhancement of bio-hydrogen production.

Bio-hydrogen production from dry thermophilic anaerobic co-digestion (55°C and 20% total solids) of organic fraction of municipal solid waste (OFMSW) and food waste (FW) was studied. OFMSW coming from mechanical-biological treatment plants (MBT plants) presents a low organic matter concentration. However, FW has a high organic matter content but several problems by accumulation of volatile fatty acids (VFAs) and system acidification. Tests were conducted using a mixture ratio of 80:20 (OFSMW:FW), to avoid the aforementioned problems. Different solid retention times (SRTs) - 6.6, 4.4, 2.4 and 1.9 days - were tested. It was noted that addition of food waste enhances the hydrogen production in all the SRTs tested. Best results were obtained at 1.9-day SRT. It was observed an increase from 0.64 to 2.51 L H2/L(reactor) day in hydrogen productivity when SRTs decrease from 6.6 to 1.9 days. However, the hydrogen yield increases slightly from 33.7 to 38 mL H2/gVS(added).

Next-Generation Sequencing Panels for the Diagnosis of Colorectal Cancer and Polyposis Syndromes: A Cost-Effectiveness Analysis.

PURPOSE: To evaluate the cost effectiveness of next-generation sequencing (NGS) panels for the diagnosis of colorectal cancer and polyposis (CRCP) syndromes in patients referred to cancer genetics clinics. PATIENTS AND METHODS: We developed a decision model to evaluate NGS panel testing compared with current standard of care in patients referred to a cancer genetics clinic. We obtained data on the prevalence of genetic variants from a large academic laboratory and calculated the costs and health benefits of identifying relatives with a pathogenic variant, in life-years and quality-adjusted life-years (QALYs). We classified the CRCP syndromes according to their type of inheritance and penetrance of colorectal cancer. One-way and probabilistic sensitivity analyses were conducted to assess uncertainty. RESULTS: Evaluation with an NGS panel that included Lynch syndrome genes and other genes associated with highly penetrant CRCP syndromes led to an average increase of 0.151 year of life, 0.128 QALY, and $4,650 per patient, resulting in an incremental cost-effectiveness ratio of $36,500 per QALY compared with standard care and a 99% probability that this panel was cost effective at a threshold of $100,000 per QALY. When compared with this panel, the addition of genes with low colorectal cancer penetrance resulted in an incremental cost-effectiveness ratio of $77,300 per QALY. CONCLUSION: The use of an NGS panel that includes genes associated with highly penetrant CRCP syndromes in addition to Lynch syndrome genes as a first-line test is likely to provide meaningful clinical benefits in a cost-effective manner at a $100,000 per QALY threshold.

Actionable exomic incidental findings in 6503 participants: challenges of variant classification.

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

The cost-effectiveness of returning incidental findings from next-generation genomic sequencing.

PURPOSE: The American College of Medical Genetics and Genomics (ACMG) recommended that clinical laboratories performing next-generation sequencing analyze and return pathogenic variants for 56 specific genes it considered medically actionable. Our objective was to evaluate the clinical and economic impact of returning these results. METHODS: We developed a decision-analytic policy model to project the quality-adjusted life-years and lifetime costs associated with returning ACMG-recommended incidental findings in three hypothetical cohorts of 10,000 patients. RESULTS: Returning incidental findings to cardiomyopathy patients, colorectal cancer patients, or healthy individuals would increase costs by $896,000, $2.9 million, and $3.9 million, respectively, and would increase quality-adjusted life-years by 20, 25.4, and 67 years, respectively, for incremental cost-effectiveness ratios of $44,800, $115,020, and $58,600, respectively. In probabilistic analyses, returning incidental findings cost less than $100,000/quality-adjusted life-year gained in 85, 28, and 91%, respectively, of simulations. Assuming next-generation sequencing costs $500, the incremental cost-effectiveness ratio for primary screening of healthy individuals was $133,400 (<$100,000/quality-adjusted life-year gained in 10% of simulations). Results were sensitive to the cohort age and assumptions about gene penetrance. CONCLUSION: Returning incidental findings is likely cost-effective for certain patient populations. Screening of generally healthy individuals is likely not cost-effective based on current data, unless next-generation sequencing costs less than $500.

Comparative effectiveness of next generation genomic sequencing for disease diagnosis: design of a randomized controlled trial in patients with colorectal cancer/polyposis syndromes.

Whole exome and whole genome sequencing are applications of next generation sequencing transforming clinical care, but there is little evidence whether these tests improve patient outcomes or if they are cost effective compared to current standard of care. These gaps in knowledge can be addressed by comparative effectiveness and patient-centered outcomes research. We designed a randomized controlled trial that incorporates these research methods to evaluate whole exome sequencing compared to usual care in patients being evaluated for hereditary colorectal cancer and polyposis syndromes. Approximately 220 patients will be randomized and followed for 12 months after return of genomic findings. Patients will receive findings associated with colorectal cancer in a first return of results visit, and findings not associated with colorectal cancer (incidental findings) during a second return of results visit. The primary outcome is efficacy to detect mutations associated with these syndromes; secondary outcomes include psychosocial impact, cost-effectiveness and comparative costs. The secondary outcomes will be obtained via surveys before and after each return visit. The expected challenges in conducting this randomized controlled trial include the relatively low prevalence of genetic disease, difficult interpretation of some genetic variants, and uncertainty about which incidental findings should be returned to patients. The approaches utilized in this study may help guide other investigators in clinical genomics to identify useful outcome measures and strategies to address comparative effectiveness questions about the clinical implementation of genomic sequencing in clinical care.

Return of genomic results to research participants: the floor, the ceiling, and the choices in between.

As more research studies incorporate next-generation sequencing (including whole-genome or whole-exome sequencing), investigators and institutional review boards face difficult questions regarding which genomic results to return to research participants and how. An American College of Medical Genetics and Genomics 2013 policy paper suggesting that pathogenic mutations in 56 specified genes should be returned in the clinical setting has raised the question of whether comparable recommendations should be considered in research settings. The Clinical Sequencing Exploratory Research (CSER) Consortium and the Electronic Medical Records and Genomics (eMERGE) Network are multisite research programs that aim to develop practical strategies for addressing questions concerning the return of results in genomic research. CSER and eMERGE committees have identified areas of consensus regarding the return of genomic results to research participants. In most circumstances, if results meet an actionability threshold for return and the research participant has consented to return, genomic results, along with referral for appropriate clinical follow-up, should be offered to participants. However, participants have a right to decline the receipt of genomic results, even when doing so might be viewed as a threat to the participants' health. Research investigators should be prepared to return research results and incidental findings discovered in the course of their research and meeting an actionability threshold, but they have no ethical obligation to actively search for such results. These positions are consistent with the recognition that clinical research is distinct from medical care in both its aims and its guiding moral principles.

Actionable, pathogenic incidental findings in 1,000 participants' exomes.

The incorporation of genomics into medicine is stimulating interest on the return of incidental findings (IFs) from exome and genome sequencing. However, no large-scale study has yet estimated the number of expected actionable findings per individual; therefore, we classified actionable pathogenic single-nucleotide variants in 500 European- and 500 African-descent participants randomly selected from the National Heart, Lung, and Blood Institute Exome Sequencing Project. The 1,000 individuals were screened for variants in 114 genes selected by an expert panel for their association with medically actionable genetic conditions possibly undiagnosed in adults. Among the 1,000 participants, 585 instances of 239 unique variants were identified as disease causing in the Human Gene Mutation Database (HGMD). The primary literature supporting the variants' pathogenicity was reviewed. Of the identified IFs, only 16 unique autosomal-dominant variants in 17 individuals were assessed to be pathogenic or likely pathogenic, and one participant had two pathogenic variants for an autosomal-recessive disease. Furthermore, one pathogenic and four likely pathogenic variants not listed as disease causing in HGMD were identified. These data can provide an estimate of the frequency (∼3.4% for European descent and ∼1.2% for African descent) of the high-penetrance actionable pathogenic or likely pathogenic variants in adults. The 23 participants with pathogenic or likely pathogenic variants were disproportionately of European (17) versus African (6) descent. The process of classifying these variants underscores the need for a more comprehensive and diverse centralized resource to provide curated information on pathogenicity for clinical use to minimize health disparities in genomic medicine.

The Electronic Medical Records and Genomics (eMERGE) Network: past, present, and future.

The Electronic Medical Records and Genomics Network is a National Human Genome Research Institute-funded consortium engaged in the development of methods and best practices for using the electronic medical record as a tool for genomic research. Now in its sixth year and second funding cycle, and comprising nine research groups and a coordinating center, the network has played a major role in validating the concept that clinical data derived from electronic medical records can be used successfully for genomic research. Current work is advancing knowledge in multiple disciplines at the intersection of genomics and health-care informatics, particularly for electronic phenotyping, genome-wide association studies, genomic medicine implementation, and the ethical and regulatory issues associated with genomics research and returning results to study participants. Here, we describe the evolution, accomplishments, opportunities, and challenges of the network from its inception as a five-group consortium focused on genotype-phenotype associations for genomic discovery to its current form as a nine-group consortium pivoting toward the implementation of genomic medicine.

Genetic variation associated with circulating monocyte count in the eMERGE Network.

With white blood cell count emerging as an important risk factor for chronic inflammatory diseases, genetic associations of differential leukocyte types, specifically monocyte count, are providing novel candidate genes and pathways to further investigate. Circulating monocytes play a critical role in vascular diseases such as in the formation of atherosclerotic plaque. We performed a joint and ancestry-stratified genome-wide association analyses to identify variants specifically associated with monocyte count in 11 014 subjects in the electronic Medical Records and Genomics Network. In the joint and European ancestry samples, we identified novel associations in the chromosome 16 interferon regulatory factor 8 (IRF8) gene (P-value = 2.78×10(-16), ß = -0.22). Other monocyte associations include novel missense variants in the chemokine-binding protein 2 (CCBP2) gene (P-value = 1.88×10(-7), ß = 0.30) and a region of replication found in ribophorin I (RPN1) (P-value = 2.63×10(-16), ß = -0.23) on chromosome 3. The CCBP2 and RPN1 region is located near GATA binding protein2 gene that has been previously shown to be associated with coronary heart disease. On chromosome 9, we found a novel association in the prostaglandin reductase 1 gene (P-value = 2.29×10(-7), ß = 0.16), which is downstream from lysophosphatidic acid receptor 1. This region has previously been shown to be associated with monocyte count. We also replicated monocyte associations of genome-wide significance (P-value = 5.68×10(-17), ß = -0.23) at the integrin, alpha 4 gene on chromosome 2. The novel IRF8 results and further replications provide supporting evidence of genetic regions associated with monocyte count.

Use of array comparative genome hybridization in orofacial clefting.

Orofacial clefting is a common condition found in 1 per 700 to 1 per 1000 births. Although most cases are isolated, a subset is caused by a specific genetic mutation. Specific gene tests have been used for recognizable syndromes such as velocardiofacial syndrome or van der Woude syndrome, where the cleft is associated with other anomalies. However, many cleft lip and palate patients have other anomalies but do not fit in to a recognizable syndrome. For these patients, chromosome analysis has been a first-line genetic test; however, in the past few years, a new form of genetic testing has become available for these patients: array comparative genome hybridization (aCGH). We present a 7-month-old male infant with cleft palate, developmental delay, and a family history of velopharyngeal insufficiency in whom aCGH array was used to identify a small deletion on the short (p) arm of chromosome 7. This defect, which was also found in the mother, was undetected by chromosome analysis. In summary, this case demonstrates that aCGH is a new diagnostic tool that is useful in the evaluation of select cases of orofacial clefting. Array comparative genome hybridization should be considered when the suspicion for a genetic etiology of the clefting remains strong despite a normal cytogenetic analysis.

Cardiac-restricted angiotensin-converting enzyme overexpression causes conduction defects and connexin dysregulation.

Renin-angiotensin (RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrio-Hisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na(+) channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca(2+) channel, Kv4.2 K(+) channel alpha-subunit, and Cx45 mRNA abundances and the peak ventricular Na(+) current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.