Continued lessons from the INS gene: An intronic mutation causing diabetes through a novel mechanism

Background Diabetes in neonates usually has a monogenic aetiology; however, the cause remains unknown in 20-30%. Heterozygous INS mutations represent one of the most common gene causes of neonatal diabetes mellitus. Methods Clinical and functional characterisation of a novel homozygous intronic mutation (c.187+241G>A) in the insulin gene in a child identified through the Monogenic Diabetes Registry (http://monogenicdiabetes. uchicago.edu). Results The proband had insulin-requiring diabetes from birth. Ultrasonography revealed a structurally normal pancreas and C-peptide was undetectable despite readily detectable amylin, suggesting the presence of dysfunctional ß cells. Whole-exome sequencing revealed the novel mutation. In silico analysis predicted a mutant mRNA product resulting from preferential recognition of a newly created splice site. Wild-type and mutant human insulin gene constructs were derived and transiently expressed in INS-1 cells. We confirmed the predicted transcript and found an additional transcript created via an ectopic splice acceptor site. Conclusions Dominant INS mutations cause diabetes via a mutated translational product causing endoplasmic reticulum stress. We describe a novel mechanism of diabetes, without ß cell death, due to creation of two unstable mutant transcripts predicted to undergo nonsense and non-stop-mediated decay, respectively. Our discovery may have broader implications for those with insulin deficiency later in life

The dynamic use of EGFR mutation analysis in cell-free DNA as a follow-up biomarker during different treatment lines in non-small-cell lung cancer patients

Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median = 11, IQR = 9 5-13) to the best response (median = 0, IQR = 0-0, p < 0 01), followed by a significant increase at progression (median = 11, IQR = 11-15, p < 0 01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received

Next-generation sequencing improves precision medicine in hearing loss

Background: An early etiological diagnosis of hearing loss positively impacts children’s quality of life including language and cognitive development. Even though hearing loss associates with extremely high genetic and allelic heterogeneity, several studies have proven that Next-Generation Sequencing (NGS)-based gene panel testing significantly reduces the time between onset and diagnosis.Methods: In order to assess the clinical utility of our custom NGS GHELP panel, the prevalence of pathogenic single nucleotide variants, indels or copy number variants was assessed by sequencing 171 nuclear and 8 mitochondrial genes in 155 Spanish individuals with hearing loss.Results: A genetic diagnosis of hearing loss was achieved in 34% (52/155) of the individuals (5 out of 52 were syndromic). Among the diagnosed cases, 87% (45/52) and 12% (6/52) associated with autosomal recessive and dominant inheritance patterns respectively; remarkably, 2% (1/52) associated with mitochondrial inheritance pattern. Although the most frequently mutated genes in this cohort were consistent with those described in the literature (GJB2, OTOF or MYO7A), causative variants in less frequent genes such as TMC1, FGF3 or mitCOX1 were also identified. Moreover, 5% of the diagnosed cases (3/52) were associated with pathogenic copy number variants.Conclusion: The clinical utility of NGS panels that allows identification of different types of pathogenic variants–not only single nucleotide variants/indels in both nuclear and mitochondrial genes but also copy number variants–has been demonstrated to reduce the clinical diagnostic odyssey in hearing loss. Thus, clinical implementation of genomic strategies within the regular clinical practice, and, more significantly, within the newborn screening protocols, is warranted

Does arterial hypertension influence the onset of Huntington's disease?

Neurological Motor Disorder

Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures

Objective: despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). Design: a prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. Results: an initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. Conclusion: implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.Funding: we thank the financial support of CIBERehd; grants PI16/01126 and PI19/00163 from Instituto de Salud Carlos III (ISCIII) cofinanced by ’Fondo Europeo de Desarrollo Regional’ (FEDER) ’Una manera de hacer Europa’; grants 58/2017 and 55/2018 from Gobierno de Navarra Salud; grant 0011-1411-2020-000010 from AGATA Strategic Project from Gobierno de Navarra; grant 2020/101 from Euroregion Nouvelle Aquitaine-Euskadi-Navarra; Fundación Eugenio Rodríguez Pascual; Fundación Mario Losantos, Fundación M Torres; grant 2018/117 from AMMF, the Cholangiocarcinoma Charity; the COST Action CA181122 Euro-cholangio-Net; POSTD18014AREC postdoctoral fellowship from AECC to MA; and Ramón y Cajal Program contracts RYC-2014-15242 and RYC-2018-024475-1 to FJC and MGFB

Adaptations to Climate-Mediated Selective Pressures in Humans

Humans inhabit a remarkably diverse range of environments, and adaptation through natural selection has likely played a central role in the capacity to survive and thrive in extreme climates. Unlike numerous studies that used only population genetic data to search for evidence of selection, here we scan the human genome for selection signals by identifying the SNPs with the strongest correlations between allele frequencies and climate across 61 worldwide populations. We find a striking enrichment of genic and nonsynonymous SNPs relative to non-genic SNPs among those that are strongly correlated with these climate variables. Among the most extreme signals, several overlap with those from GWAS, including SNPs associated with pigmentation and autoimmune diseases. Further, we find an enrichment of strong signals in gene sets related to UV radiation, infection and immunity, and cancer. Our results imply that adaptations to climate shaped the spatial distribution of variation in humans

Hypoxia, AMPK activation and uterine artery vasoreactivity

This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1113/JP270995Genes near adenosine monophosphate-activated protein kinase-α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude-adapted Andean populations. Adenosine monophosphate-activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near-term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7-9) from day 14 to day 19, and placentas from non-labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near-term murine UtA and placental tissue. RT-PCR products for AMPK-α1 and -α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK-α1 and -α2 expression in the high- compared with low-altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre-constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l-NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO-dependent and NO-independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero-placental blood flow in hypoxic pregnancy.Funding for these studies was provided by the Wellcome Trust (084804/2/08/Z) to G.J.B., the British Heart Foundation and the Wellcome Trust to D.A.G., the Biotechnology and Biological Sciences Research Council (BBSRC) to A.L.F., a UK Wellcome Trust Programme Grant (WT081195MA) to A.M.E. and A.D.M., a BBSRC studentship and in vivo skills award to J.S.H., a National Health Medical Research Council and Centre for Trophoblast Research fellowship to A.N.S.-P., and a NIH RO1 grant (HLBI-079647) to L.G.M. along with sabbatical support from Wake Forest University

Familial primary cutaneous amyloidosis: Caspase activation may be involved in amyloid formation

Primary localized cutaneous amyloidosis (PLCA) is a rare form of cutaneous amyloidosis, characterized by the presence of flat-topped papules and macules with amyloid deposits in the superficial dermis. It is a purely cutaneous disease with no association with systemic forms of amyloidosis.1 Although most cases are sporadic, familial cases (FPLCA) represent about 10% of total reports and show an autosomal dominant inheritance, with mutations described in the genes for the oncostatin M receptor (OSMR) and the interleukin-31 receptor A (IL31RA).2 Herein, we present a family affected by FPLCA and underline the role of caspase activation in amyloid formation