Novel Pathogenic Variants Leading to Sporadic Amyotrophic Lateral Sclerosis in Greek Patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease that affects motor neurons, leading to paralysis and death usually 3-5 years after the onset of symptoms. The investigation of both sporadic and familial ALS highlighted four main genes that contribute to the pathogenesis of the disease: SOD1, FUS, TARDBP and C9orf72. This study aims to provide a comprehensive investigation of genetic variants found in SOD1, FUS and TARDBP genes in Greek sporadic ALS (sALS) cases. Our sequencing analysis of the coding regions of the abovementioned genes that include the majority of the variants that lead to ALS in 32 sALS patients and 3 healthy relatives revealed 6 variants in SOD1, 19 variants in FUS and 37 variants in TARDBP, of which the SOD1 p.D90A and the FUS c.*356G>A (rs886051940) variants have been previously associated with ALS, while two novel nonsense pathogenic variants were also identified, namely FUS p.R241* and TDP-43 p.Y214*. Our study contributes to the worldwide effort toward clarifying the genetic basis of sALS to better understand the disease's molecular pathology.

Assessment of pressure injury knowledge using PZ-PUKT questionnaire among Indian nurses and the evaluation of impact of an educational intervention on the knowledge scores: A quasi-experimental study.

AIM: To determine the knowledge of pressure injury among Indian nurses using PZ-PUKT questionnaire and to evaluate the effect of an educational intervention on knowledge scores. MATERIALS AND METHODS: A Quasi-experimental study design was used to test the Pressure injury knowledge of 273 nurses in a tertiary care teaching hospital. The Pieper Zulkowski Pressure Ulcer Knowledge Test questionnaire was given as a pre-test prior to education session and re-administered after the activity was completed. RESULTS: The mean score of the pre-test was 48.58 ± 6.75 (C·I 47.8-49.4) and post-test 54.14 ± 7.69 (C·I 53.3-55.1), which showed a statistically significant improvement. In the pre-test, nurses had highest score in the prevention subset while wound subset had the greatest improvement in the post-test. Female participants had a better understanding about Pressure injuries when compared to males. Also, the Assistant Nursing Superintendents and ward in Charge nurses had better knowledge as compared with other nurses. CONCLUSIONS: The knowledge of pressure injury among nurses is limited. Knowledge deficits should be identified and targeted educational interventions should be administered to all the nurses irrespective of their educational level and work experience. Wound certification courses should be instituted so that it gives the nurses a better opportunity to learn about pressure injuries at a certified level. All nurses should undergo periodic training in this ever-evolving field so as to provide the best care to their patients.

Spectrum of genetic variants in bilateral sensorineural hearing loss.

Background: Hearing loss (HL) is an impairment of auditory function with identified genetic forms that can be syndromic (30%) or non-syndromic (70%). HL is genetically heterogeneous, with more than 1,000 variants across 150 causative genes identified to date. The genetic diagnostic rate varies significantly depending on the population being tested. Countries with a considerably high rate of consanguinity provide a unique resource for studying rare forms of recessive HL. In this study, we identified genetic variants associated with bilateral sensorineural HL (SNHL) using whole-exome sequencing (WES) in 11 families residing in the United Arab Emirates (UAE). Results: We established the molecular diagnosis in six probands, with six different pathogenic or likely pathogenic variants in the genes MYO15A, SLC26A4, and GJB2. One novel nonsense variant, MYO15A:p.Tyr1962Ter*, was identified in a homozygous state in one family, which has not been reported in any public database. SLC26A4 and GJB2 were found to be the most frequently associated genes in this study. In addition, six variants of uncertain significance (VUS) were detected in five probands in the genes CDH23, COL11A1, ADGRV1, NLRP3, and GDF6. In total, 12 variants were observed in eight genes. Among these variants, eight missense variants (66.7%), three nonsense variants (25.0%), and one frameshift (8.3%) were identified. The overall diagnostic rate of this study was 54.5%. Approximately 45.5% of the patients in this study came from consanguineous families. Conclusion: Understanding the genetic basis of HL provides insight for the clinical diagnosis of hearing impairment cases through the utilization of next-generation sequencing (NGS). Our findings contribute to the knowledge of the heterogeneous genetic profile of HL, especially in a population with a high rate of consanguineous marriage in the Arab population.

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature.

Introduction: Natriuretic peptide receptor 2 (NPR2 or NPR-B) plays a central role in growth development and bone morphogenesis and therefore loss-of-function variations in NPR2 gene have been reported to cause Acromesomelic Dysplasia, Maroteaux type 1 and short stature. While several hypotheses have been proposed to underlie the pathogenic mechanisms responsible for these conditions, the exact mechanisms, and functional characteristics of many of those variants and their correlations with the clinical manifestations have not been fully established. Methods: In this study, we examined eight NPR2 genetic missense variants (p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg318Gly, p.Arg388Gln, p.Arg495Cys, p.Arg557His, and p.Arg932Cys) Acromesomelic Dysplasia, Maroteaux type 1 and short stature located on diverse domains and broadly classified as variants of uncertain significance. The evaluated variants are either reported in patients with acromesomelic dysplasia in the homozygous state or short stature in the heterozygous state. Our investigation included the evaluation of their expression, subcellular trafficking and localization, N-glycosylation profiles, and cyclic guanosine monophosphate (cGMP) production activity. Results and Discussion: Our results indicate that variants p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg388Gln have defective cellular trafficking, being sequestered within the endoplasmic reticulum (ER), and consequently impaired cGMP production ability. Conversely, variants p.Arg318Gly, p.Arg495Cys, and p.Arg557His seem to display a non-statistically significant behavior that is slightly comparable to WT-NPR2. On the other hand, p.Arg932Cys which is located within the guanylyl cyclase active site displayed normal cellular trafficking profile albeit with defective cGMP. Collectively, our data highlights the genotype-phenotype relationship that might be responsible for the milder symptoms observed in short stature compared to acromesomelic dysplasia. This study enhances our understanding of the functional consequences of several NPR2 variants, shedding light on their mechanisms and roles in related genetic disorders which might also help in their pathogenicity re-classification.

Case report: Birk-Landau-Perez syndrome linked to the SLC30A9 gene-identification of additional cases and expansion of the phenotypic spectrum.

Birk-Landau-Perez syndrome (BILAPES) is an autosomal recessive cerebro-renal syndrome associated with genetic defects in the SLC30A9 gene, initially reported in 2017 in six individuals belonging to a large Bedouin kindred. The SLC30A9 gene encodes a putative mitochondrial zinc transporter with ubiquitous expression, the highest found in the brain, kidney, and skeletal muscle. Since the first report, only one additional affected patient has been described, but there were some inconsistencies, such as hearing loss, failure to thrive, and neuroimaging findings between the clinical presentation of the disease in the Bedouin family and the second patient. Here, we present two more patients from a consanguineous Middle Eastern family with features of chronic kidney disease, neurodevelopmental regression, ataxia, hearing loss, and eye abnormalities, which were largely consistent with BILAPES. Whole-exome sequencing detected a homozygous in-frame deletion c.1049_1051delCAG (p.Ala350del) in the SLC30A9 gene, which was the same variant detected in the patients from the primary literature report and the variant segregated with disease in the family. However, in the patients described here, brain MRI showed cerebellar atrophy, which was not a cardinal feature of the syndrome from the primary report. Our findings provide further evidence for SLC30A9-associated BILAPES and contribute to defining the clinical spectrum.

Novel compound heterozygous variants (c.971delA/c.542C > T) in SLC1A4 causes spastic tetraplegia, thin corpus callosum, and progressive microcephaly: a case report and mutational analysis.

Spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) are linked to SLC1A4 genetic variants since the first reported case in 2015. SLC1A4 encodes for the neutral amino acid transporter ASCT1 which is involved in the transportation of serine between astrocytes and neurons. Although most of the reported cases are of Ashkenazi Jewish ancestry, SPATCCM has also been reported in Irish, Italian, Czech, Palestinian, and Pakistani ethnicities. Herein, we report two Pakistani male siblings from a non-consanguineous marriage presented with global developmental delay associated with spastic quadriplegia, microcephaly, and infantile spasm. Since infancy, both siblings suffered from microcephaly with brain MRI demonstrating generalized atrophy of the frontal, temporal, and parietal lobes with a prominence of the subarachnoid spaces, widening of the Sylvian fissures, and enlargement of the ventricular system not compatible with the chronological age of both patients associated with thinning of the corpus callosum. Whole-exome sequencing of both affected brothers revealed novel compound heterozygous variants in the SLC1A4 gene (NM_003038) segregating from their parents. The maternal c.971delA (p.N324Tfs*29) deletion variant disturbs the transcript reading frame leading to the generation of a premature stop codon and its subsequent degradation by nonsense-mediated mRNA decay as detected through expression analysis. The paternal c.542C > T (p.S181F) missense variant was predicted deleterious via multiple in silico prediction tools as the amino acid substitution is speculated to affect the overall ASCT1 structural confirmation due to the loss of an H-bond at the core of the protein at this position which might affect its function as concluded from the simulation analysis. The presented cases expand the genetic and clinical spectrum of ASCT1 deficiency and support the importance of including SLC1A4 gene screening in infants with unexplained global neurodevelopmental delay regardless of ethnicity.

GRP78/BiP alleviates oxLDL-induced hepatotoxicity in familial hypercholesterolemia caused by missense variants of LDLR in a HepG2 cellular model.

BACKGROUND AND AIMS: The accumulation of misfolded proteins, encoded by genetic variants of functional genes leads to Endoplasmic Reticulum (ER) stress, which is a critical consequence in human disorders such as familial hypercholesterolemia, cardiovascular and hepatic diseases. In addition to the identification of ER stress as a contributing factor to pathogenicity, extensive studies on the role of oxidized Low-Density Lipoprotein (oxLDL) and its ill effects in expediting cardiovascular diseases and other metabolic comorbidities are well documented. However, the current understanding of its role in hepatic insults needs to be revised. This study elucidates the molecular mechanisms underlying the progression of oxLDL and ER stress-induced cytotoxicity in HepG2. METHODS: HepG2 cells stably expressing wild-type Low-Density lipoprotein receptor (WT-LDLR) and missense variants of LDLR that are pathogenically associated with familial hypercholesterolemia were used as the in vitro models. The relative mRNA expression and protein profiles of ER stress sensors, inflammatory and apoptotic markers, together with cytotoxic assays and measurement of mitochondrial membrane potential, were carried out in HepG2 cells treated with 100 µg per ml oxLDL for 24 to 48 h. 1-way or 2-way ANOVA was used for statistical analyses of datasets. RESULTS: ER stress responses are elicited along all three arms of the unfolded protein response (UPR), with adverse cytotoxic and inflammatory responses in oxLDL-treated conditions. Interestingly, oxLDL-treated ER-stressed HepG2 cells manifested intriguingly low expression of BiP- the master regulator of ER stress, as observed earlier by various researchers in liver biopsies of Non-Alcoholic Steatohepatitis (NASH) patients. This study shows that overexpression of BiP rescues hepatic cells from cytotoxic and inflammatory mechanisms instigated by ER stress in combination with oxLDL, along the ER and mitochondrial membrane and restores cellular homeostasis. CONCLUSION: The data provide interesting leads that identify patients with familial hypercholesterolemia conditions and potentially other Endoplasmic Reticulum Associated Degradation (ERAD) diseases as highly susceptible to developing hepatic insults with molecular signatures like those manifested in Non-Alcoholic Fatty Liver Disease (NAFLD) and NASH. LIMITATIONS AND FUTURE PERSPECTIVES: Although the use of HepG2 cells as the model is a major caveat of the study, the findings of this research may be used as the pilot study to expand further investigations in primary hepatocytes or iPSC- derived cellular models.

Characterization of ACE2 naturally occurring missense variants: impact on subcellular localization and trafficking.

BACKGROUND: Human angiotensin-converting enzyme 2 (ACE2), a type I transmembrane receptor physiologically acting as a carboxypeptidase enzyme within the renin-angiotensin system (RAS), is a critical mediator of infection by several severe acute respiratory syndrome (SARS) corona viruses. For instance, it has been demonstrated that ACE2 is the primary receptor for the SARS-CoV-2 entry to many human cells through binding to the viral spike S protein. Consequently, genetic variability in ACE2 gene has been suggested to contribute to the variable clinical manifestations in COVID-19. Many of those genetic variations result in missense variants within the amino acid sequence of ACE2. The potential effects of those variations on binding to the spike protein have been speculated and, in some cases, demonstrated experimentally. However, their effects on ACE2 protein folding, trafficking and subcellular targeting have not been established. RESULTS: In this study we aimed to examine the potential effects of 28 missense variants (V801G, D785N, R768W, I753T, L731F, L731I, I727V, N720D, R710H, R708W, S692P, E668K, V658I, N638S, A627V, F592L, G575V, A501T, I468V, M383I, G173S, N159S, N149S, D38E, N33D, K26R, I21T, and S19P) distributed across the ACE2 receptor domains on its subcellular trafficking and targeting through combinatorial approach involving in silico analysis and experimental subcellular localization analysis. Our data show that none of the studied missense variants (including 3 variants predicted to be deleterious R768W, G575V, and G173S) has a significant effect on ACE2 intracellular trafficking and subcellular targeting to the plasma membrane. CONCLUSION: Although the selected missense variants display no significant change in ACE2 trafficking and subcellular localization, this does not rule out their effect on viral susceptibility and severity. Further studies are required to investigate the effect of ACE2 variants on its expression, binding, and internalization which might explain the variable clinical manifestations associated with the infection.

Endoglin Wild Type and Variants Associated With Hereditary Hemorrhagic Telangiectasia Type 1 Undergo Distinct Cellular Degradation Pathways.

Endoglin, also known as cluster of differentiation 105 (CD105), is an auxiliary receptor in the TGFß signaling pathway. It is predominantly expressed in endothelial cells as a component of the heterotetrameric receptor dimers comprising type I, type II receptors and the binding ligands. Mutations in the gene encoding Endoglin (ENG) have been associated with hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant inherited disease that is generally characterized by vascular malformation. Secretory and many endomembrane proteins synthesized in the Endoplasmic reticulum (ER) are subjected to stringent quality control mechanisms to ensure that only properly folded and assembled proteins are trafficked forward through the secretory pathway to their sites of action. We have previously demonstrated that some Endoglin variants causing HHT1 are trapped in the ER and fail to traffic to their normal localization in plasma membrane, which suggested the possible involvement of ER associated protein degradation (ERAD) in their molecular pathology. In this study, we have investigated, for the first time, the degradation routes of Endoglin wild type and two mutant variants, P165L and V105D, and previously shown to be retained in the ER. Stably transfected HEK293 cells were treated with proteasomal and lysosomal inhibitors in order to elucidate the exact molecular mechanisms underlying the loss of function phenotype associated with these variants. Our results have shown that wild type Endoglin has a relatively short half-life of less than 2 hours and degrades through both the lysosomal and proteasomal pathways, whereas the two mutant disease-causing variants show high stability and predominantly degrades through the proteasomal pathway. Furthermore, we have demonstrated that Endoglin variants P165L and V105D are significantly accumulated in HEK293 cells deficient in HRD1 E3 ubiquitin ligase; a major ERAD component. These results implicate the ERAD mechanism in the pathology of HHT1 caused by the two variants. It is expected that these results will pave the way for more in-depth research studies that could provide new windows for future therapeutic interventions.

Spondylometaepiphyseal Dysplasia Short Limb-Abnormal Calcification Type in Turkish Patients Reveals a Novel Mutation and New Features.

Spondylometaepiphyseal dysplasia short limb-abnormal calcification type (SMED-SL/AC) is a rare autosomal recessive disorder. It is a severe dwarfism syndrome with a characteristic feature of progressive calcification of epiphyseal and other cartilaginous tissues. It is caused by pathogenic variants in the DDR2 gene encoding the discoidin domain receptor tyrosine kinase 2. Thus far, 37 cases and 8 pathogenic variants have been reported. Most of the reported cases are of Middle Eastern and Puerto Rican origins. Only one Turkish case has been reported previously with a novel truncating variant p.(R489*). Here, we report 2 new cases, 1 with a novel variant p.(S311G) and 1 with a splice site variant c.2283+1G>A. In addition, we reviewed a previously reported case, and sequencing of stored DNA revealed the recently reported nonsense variant p.(R489*) as the underlying cause. Therefore, our data increase the number of SMED-SL/AC Turkish patients with molecular results to 4. Furthermore, we compared the features of Turkish patients with other reported cases and expanded the characteristics of the disorder with new features such as triventricular hydrocephalus, intracranial hemorrhage, hypopigmentation of hair, dry and scaly skin, arthralgia, and hypocalcemia. We also compared the pathogenic variants of Turkish patients with other variants, aiming to explain the mechanism leading to a more severe and early fatal course in Turkish patients.

Bi-allelic null variant in matrix metalloproteinase-15, causes congenital cardiac defect, cholestasis jaundice, and failure to thrive.

Here, we delineate the phenotype of two siblings with a bi-allelic frameshift variant in MMP15 gene with congenital cardiac defects, cholestasis, and dysmorphism. Genome sequencing analysis revealed a recently reported homozygous frameshift variant (c.1058delC, p.Pro353Glnfs*102) in MMP15 gene that co-segregates with the phenotype in the family in a recessive mode of inheritance. Relative quantification of MMP15 mRNA showed evidence of degradation of the mutated transcript, presumably by nonsense mediated decay. Likewise, MMP15: p.Gly231Arg, a concurrently reported homozygous missense variant in another patient exhibiting a similar phenotype, was predicted to disrupt zinc ion binding to the MMP-15 enzyme catalytic domain, which is essential for substrate proteolysis, by structural modeling. Previous animal models and cellular findings suggested that MMP15 plays a crucial role in the formation of endocardial cushions. These findings confirm that MMP15 is an important gene in human development, particularly cardiac, and that its loss of function is likely to cause a severe disorder phenotype.

Unilateral pulmonary vein atresia presenting with recurrent haemoptysis in a child: a case report.

BACKGROUND: Haemoptysis is an uncommon presenting symptom in children and is usually caused by acute lower respiratory tract infection or foreign body aspiration. We report a rare case of right unilateral pulmonary vein atresia (PVA) as the underlying aetiology of recurrent haemoptysis in a child. CASE PRESENTATION: A 4 years old girl presented with history of recurrent haemoptysis. Bronchoscopic evaluation excluded a foreign body aspiration but revealed right bronchial mucosal hyperaemia and varices. Diagnosis of right unilateral PVA was suspected on transthoracic echocardiography which demonstrated hypoplastic right pulmonary artery and non-visualization of right pulmonary veins. Final diagnosis was confirmed on cardiac CT angiography. A conservative treatment approach was opted with consideration for pneumonectomy in future when she is older. CONCLUSION: Rarer causes should be considered when investigating for recurrent haemoptysis in children. Bronchoscopy and cardiac imaging are useful tools to establish the diagnosis of unilateral PVA in our case.

Multiomics Analysis Coupled with Text Mining Identify Novel Biomarker Candidates for Recurrent Cardiovascular Events.

Recurrent cardiovascular events remain an enigma that accounts for >30% of deaths worldwide. While heredity and human genetics variation play a key role, host-environment interactions offer a sound conceptual framework to dissect the molecular basis of recurrent cardiovascular events from genes and proteins to metabolites, thus accounting for environmental contributions as well. We report here a multiomics systems science approach so as to map interindividual variability in susceptibility to recurrent cardiovascular events. First, we performed data and text mining through a mixed-methods content analysis to select genomic variants, 10 single nucleotide polymorphisms, and microRNAs (miR-10a, miR-21, and miR-20a), minimizing bias in candidate marker selection. Next, we validated our in silico data in a patient cohort suffering from recurrent cardiovascular events (a cross-sectional study design and sampling). Our findings report a key role in low-density lipoprotein clearance for rs11206510 (p < 0.01) and rs515135 (p < 0.05). miR-10a (p < 0.05) was significantly associated with heart failure, while increased expression levels for miR-21 and miR-20a associated with atherosclerosis. In addition, liquid chromatography-mass spectrometry-based (LC-MS-based) proteomics analyses identified that vascular diameter and cholesterol levels are among the key factors to be considered in recurrent cardiovascular events. From a methodology innovation standpoint, this study offers a strategy to enhance the signal-to-noise ratios in mapping novel biomarker candidates wherein each research and conceptual step were interrogated for their validity and in turn, enriched one another, ideally translating information growth to knowledge growth.

A Novel SLC1A4 Mutation (p.Y191*) Causes Spastic Tetraplegia, Thin Corpus Callosum, and Progressive Microcephaly (SPATCCM) With Seizure Disorder.

Spastic tetraplegia, thin corpus callosum, and progressive microcephaly is a recently described very rare autosomal recessive neurodevelopmental disorder. This disease was first described in 2015 in several families from the Ashkenazi Jewish ancestry with a founder mutation in SLC1A4 (p.E256K) as the underlying genetic cause. SLC1A4 gene encodes for the amino acid transporter ASCT1 that is necessary for serine cellular transport to neurons. We clinically evaluated 2 Pakistani siblings with severe global developmental delay, progressive microcephaly, and seizure disorder. We performed exome sequencing, Sanger sequencing, and segregation analysis to identify the genetic cause of the phenotype followed by in silico analysis to evaluate the pathogenicity of the identified mutation. We identified a novel homozygous variant (c.573T>G) in both patients. The mutation is predicted to cause nonsense mutation (p.Y191*) in the ASCT1 protein. Here, we report the fifth disease causing mutation in SLC1A4 gene and review all previously reported cases.

Endoplasmic reticulum quality control of LDLR variants associated with familial hypercholesterolemia.

Loss-of-function mutations in the low-density lipoprotein receptor (LDLR) gene can cause familial hypercholesterolemia (FH), but detailed functional evidence for pathogenicity is limited to a few reported mutations. Here, we investigated the cellular pathogenic mechanisms of three mutations in LDLR causing FH, which are structurally identical to pathogenic mutations in the very low-density lipoprotein receptor (VLDLR). Similar to the VLDLR mutants, LDLR mutants D482H and C667F were found to be localized to the ER, while D445E, which is a conserved amino acid change, did not affect the trafficking of the receptor to the plasma membrane, as confirmed by the N-glycosylation profile. Although the ER-retained mutant proteins were soluble, induction of ER stress was observed as indicated by spliced X-box binding protein-1 (XBP-1) mRNA levels. The mutants were found to associate with ER quality control components, and their stability was enhanced by inhibitors of proteasome. Our results contribute to the growing list of transport-deficient class II LDLR variants leading to FH and provide evidence for the involvement of endoplasmic reticulum-associated degradation in their stability.

Genomic variants in members of the Krüppel-like factor gene family are associated with disease severity and hydroxyurea treatment efficacy in ß-hemoglobinopathies patients.

Aim: ß-Type hemoglobinopathies are characterized by vast phenotypic diversity as far as disease severity is concerned, while differences have also been observed in hydroxyurea (HU) treatment efficacy. These differences are partly attributed to the residual expression of fetal hemoglobin (HbF) in adulthood. The Krüppel-like family of transcription factors (KLFs) are a set of zinc finger DNA-binding proteins which play a major role in HbF regulation. Here, we explored the possible association of variants in KLF gene family members with response to HU treatment efficacy and disease severity in ß-hemoglobinopathies patients. Materials & methods: Six tag single nucleotide polymorphisms, located in four KLF genes, namely KLF3, KLF4, KLF9 and KLF10, were analyzed in 110 ß-thalassemia major patients (TDT), 18 nontransfusion dependent ß-thalassemia patients (NTDT), 82 sickle cell disease/ß-thalassemia compound heterozygous patients and 85 healthy individuals as controls. Results: Our findings show that a KLF4 genomic variant (rs2236599) is associated with HU treatment efficacy in sickle cell disease/ß-thalassemia compound heterozygous patients and two KLF10 genomic variants (rs980112, rs3191333) are associated with persistent HbF levels in NTDT patients. Conclusion: Our findings provide evidence that genomic variants located in KLF10 gene may be considered as potential prognostic biomarkers of ß-thalassemia clinical severity and an additional variant in KLF4 gene as a pharmacogenomic biomarker, predicting response to HU treatment.

Further Delineation of the Microcephaly-Micromelia Syndrome Associated with Loss-of-Function Variants in DONSON.

The DONSON gene encodes the downstream neighbor of SON, a replisome component that stabilizes the replication fork during replication. A severe form of microcephalic dwarfism, microcephaly-micromelia syndrome (MIMIS), has been recently associated with DONSON biallelic loss of function. Affected fetuses suffer severe growth restriction, microcephaly, and variable limb malformations which result in intrauterine or perinatal death. All described fetuses carried a homozygous founder mutation (c.1047-9A>G), a splice-altering variant that leads to transcript degradation. We evaluated 2 newborns from a consanguineous Emirati family with severe microcephaly, micromelia, craniofacial dysmorphism, and skeletal abnormalities; both died shortly after birth. Here, we report the second homozygous loss-of-function variant (c.763C>T) in DONSON causing MIMIS, and we provide detailed clinical description of this very rare disorder. In addition, we review all MIMIS cases in the literature and summarize the striking features of this phenotype. This manuscript is aimed to increase the clinical understanding of this rare, extremely severe disorder and encourage clinical and molecular geneticists to consider screening for DONSON loss-of-function variants in families with recurrent pregnancy loss and/or perinatal deaths.

Role of Genomic Biomarkers in Increasing Fetal Hemoglobin Levels Upon Hydroxyurea Therapy and in ß-Thalassemia Intermedia: A Validation Cohort Study.

Hemoglobinopathies exhibit a remarkable phenotypic diversity in terms of disease severity, while individual genetic background plays a key role in differential response to drug treatment. In the last decade, genomic variants in genes located within, as well as outside the human ß-globin cluster have been shown to be significantly associated with Hb F increase, in relation to hydroxyurea (HU) therapy in patients with these diseases. Here, we aim to determine the effect of genomic variants located in genes, such as MAP3K5, ASS1, NOS2A, TOX, PDE7B, NOS1, FLT1 and ARG2, previously shown to modulate fetal hemoglobin (Hb F) levels in patients with ß type hemoglobinopathies and reflecting disease severity and response to HU therapy in an independent cohort of Greek patients with these diseases. We recruited and genotyped 45 ß-thalassemia patients (ß-thal), either transfusion-dependent (TDT) or non transfusion-dependent (NTDT), 42 Hb S (HBB: c.20A>T)-ß-thal compound heterozygotes, who were treated with HU, as well as 53 healthy individuals, all of Hellenic origin. Our study showed that genomic variants of the MAP3K5, NOS2A and ARG2 gene are associated with HU therapy efficacy in Hb S-ß-thal compound heterozygotes. We have also shown that FLT1 and ARG2 genomic variants are associated with the mild phenotype of NTDT patients. Our findings provide evidence that MAP3K5, NOS2A, ARG2 and FLT1 genomic variants could be considered as genomic biomarkers to predict HU therapy efficacy in Hb S-ß-thal compound heterozygotes and also to describe disease severity in patients with ß type hemoglobinopathies.

Cost-effectiveness analysis of pharmacogenomics-guided clopidogrel treatment in Spanish patients undergoing percutaneous coronary intervention.

Clopidogrel is an antiplatelet drug given to patients before and after having a percutaneous coronary intervention (PCI). Genomic variants in the CYP2C19 gene are associated with variable enzyme activities affecting drug metabolism and hence, patients with reduced or increased enzymatic function have increased risk of bleeding. We conducted a cost-effectiveness analysis to compare a pharmacogenomics versus a non-pharmacogenomics-guided clopidogrel treatment for coronary artery syndrome patients undergoing PCI in the Spanish healthcare setting. A total of 549 patients diagnosed with coronary artery disease followed by PCI were recruited. Dual antiplatelet therapy was administrated to all patients from 1 to 12 months after PCI. Patients were classified into two groups: the Retrospective group was treated with clopidogrel based on the clinical routine practice and the Prospective group were initially genotyped for the presence of CYP2C19 variant alleles before treatment with those carrying more than one CYP2C19 variant alleles given prasugrel treatment. We collected data on established clinical and health outcome measures, including, per treatment arm: the percentage of patients that suffered from (a) myocardial infraction, (b) major bleeding and minor bleeding, (c) stroke, (d) the number of hospitalization days, and (e) the number of days patients spent in Intensive Care Unit. Our primary outcome measure for the cost-effectiveness analysis was Quality Adjusted Life Years (QALYs). To estimate the treatment cost for each patient, individual data on its resource used were combined with unit price data, obtained from Spanish national sources. The analysis predicts a survival of 0.9446 QALYs in the pharmacogenomics arm and 0.9379 QALYs in the non-pharmacogenomics arm within a 1-year horizon. The cumulative costs per patient were €2971 and €3205 for the Prospective and Retrospective groups, respectively. The main cost driver of total cost in both arms was hospitalization costs. The incremental cost-effectiveness ratio (ICER) was negative indicating that the PGx was a dominant option. Our data show that pharmacogenomics-guided clopidogrel treatment strategy may represent a cost-effective choice compared with non-pharmacogenomics-guided strategy for patients undergoing PCI.

DNA methylation and repressive H3K9 and H3K27 trimethylation in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and PD-L1 genes in human primary breast cancer.

Background: High expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and cancer progression. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints in cancer. Methods: Expressions of different immune checkpoint genes and PD-L1 were assessed using qRT-PCR, and the underlying epigenetic modifications including CpG methylation and repressive histone abundance were determined using bisulfite sequencing, and histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) chromatin immunoprecipitation assays (ChIP), respectively. Results: We first assessed the expression level of six immune checkpoints/ligands and found that PD-1, CTLA-4, TIM-3, and LAG-3 were significantly upregulated in breast tumor tissues (TT), compared with breast normal tissues (NT). We investigated the epigenetic modifications beyond this upregulation in immune checkpoint genes. Interestingly, we found that CpG islands in the promoter regions of PD-1, CTLA-4, and TIM-3 were significantly hypomethylated in tumor compared with normal tissues. Additionally, CpG islands of PD-L1 promoter were completely demethylated (100%), LAG-3 were highly hypomethylated (80-90%), and TIGIT were poorly hypomethylated (20-30%), in both NT and TT. These demethylation findings are in accordance with the relative expression data that, out of all these genes, PD-L1 was highly expressed and completely demethylated and TIGIT was poorly expressed and hypermethylated in both NT and TT. Moreover, bindings of H3K9me3 and H3K27me3 were found to be reduced in the promoter loci of PD-1, CTLA-4, TIM-3, and LAG-3 in tumor tissues. Conclusion: Our data demonstrate that both DNA and histone modifications are involved in upregulation of PD-1, CTLA-4, TIM-3, and LAG-3 in breast tumor tissue and these epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast cancer.