Development and Validation of a Colorectal Cancer Prediction Model: A Nationwide Cohort-Based Study.

BACKGROUND: Early diagnosis of colorectal cancer (CRC) is critical to increasing survival rates. Computerized risk prediction models hold great promise for identifying individuals at high risk for CRC. In order to utilize such models effectively in a population-wide screening setting, development and validation should be based on cohorts that are similar to the target population. AIM: Establish a risk prediction model for CRC diagnosis based on electronic health records (EHR) from subjects eligible for CRC screening. METHODS: A retrospective cohort study utilizing the EHR data of Clalit Health Services (CHS). The study includes CHS members aged 50-74 who were eligible for CRC screening from January 2013 to January 2019. The model was trained to predict receiving a CRC diagnosis within 2 years of the index date. Approximately 20,000 EHR demographic and clinical features were considered. RESULTS: The study includes 2935 subjects with CRC diagnosis, and 1,133,457 subjects without CRC diagnosis. Incidence values of CRC among subjects in the top 1% risk scores were higher than baseline (2.3% vs 0.3%; lift 8.38; P value < 0.001). Cumulative event probabilities increased with higher model scores. Model-based risk stratification among subjects with a positive FOBT, identified subjects with more than twice the risk for CRC compared to FOBT alone. CONCLUSIONS: We developed an individualized risk prediction model for CRC that can be utilized as a complementary decision support tool for healthcare providers to precisely identify subjects at high risk for CRC and refer them for confirmatory testing.

Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4.

Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole-exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2-causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/ß-catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/ß-catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain-of-function effect.

[CHARACTERIZATION OF THE INDICATIONS FOR PERFORMING GENE PANEL SEQUENCING TESTS IN A GENOMIC CENTER].

INTRODUCTION: Following the recent human genome revolution, novel technologies have been developed in the last decade enabling advanced sequencing tests, including genetic panel tests that focus on groups of specific genes associated with a certain medical condition (phenotype). Since the process of assembling a genetic panel may be complex and requires significant manpower and time, it is important to define the most common and requested panels, for gradual construction and introduction of panel tests starting with the most common. AIMS: Since no information was found in the literature defining the common panels, the aim of the study was to define the indications for performing a gene panel within the framework of the provided services, and to estimate their frequency. METHODS: Prospective data acquisition was performed by a party responsible for approval of panel tests within Clalit Health Services Organization. The indications for all approved panel tests were registered since the launch of Clalit's Genomic Center. The total number of indications was counted, and according to the Pareto principle, 20% of the most frequent indications were chosen. In addition, the indications were divided into main medical disciplines. RESULTS: Overall, 132 indications were recorded for approved gene panel tests; 20% of these indications, i.e. the first 26 indications in terms of frequency, covered 79.6% of the cases. The most frequent approved panels were epilepsy (10.4%, confidence interval (CI) 8.5-12.6%), Maturity-onset diabetes of the young (MODY) (9.6%, CI 7.8-11.7%), cardiomyopathy (8.3%, CI 6.6-10.3%) and hearing impairment (7.6%, CI 6.0-9.6%). The most common disciplines in descending order were neurological diseases (23.0%, CI 20.3-25.9%), endocrinology (13.1%, CI 11.1-15.6%), heart diseases (9.0%, CI 7.3-11.1%) and eye diseases (7.8%, CI 6.2-9.8%). CONCLUSIONS: A review of panel approvals at the Genomic Center of Clalit showed a number of frequent indications. DISCUSSION: We believe this information can be useful for the establishment of genomic laboratories, as well as for the improvement of the service to the patients, by enabling the referral for specific panel tests by medical experts who are not geneticists or genetic counselors, after appropriate training (such as the "Genetics First" program of Clalit).

Variant PADI3 in Central Centrifugal Cicatricial Alopecia.

BACKGROUND: Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry. The disease is occasionally observed to affect women in families in a manner that suggests an autosomal dominant trait and usually manifests clinically after intense hair grooming. We sought to determine whether there exists a genetic basis of CCCA and, if so, what it is. METHODS: We used exome sequencing in a group of women with alopecia (discovery set), compared the results with those in a public repository, and applied other filtering criteria to identify candidate genes. We then performed direct sequencing to identify disease-associated DNA variations and RNA sequencing, protein modeling, immunofluorescence staining, immunoblotting, and an enzymatic assay to evaluate the consequences of potential etiologic mutations. We used a replication set that consisted of women with CCCA to confirm the data obtained with the discovery set. RESULTS: In the discovery set, which included 16 patients, we identified one splice site and three heterozygous missense mutations in PADI3 in 5 patients (31%). (The approximate prevalence of the disease is up to 5.6%.) PADI3 encodes peptidyl arginine deiminase, type III (PADI3), an enzyme that post-translationally modifies other proteins that are essential to hair-shaft formation. All three CCCA-associated missense mutations in PADI3 affect highly conserved residues and are predicted to be pathogenic; protein modeling suggests that they result in protein misfolding. These mutations were found to result in reduced PADI3 expression, abnormal intracellular localization of the protein, and decreased enzymatic activity - findings that support their pathogenicity. Immunofluorescence staining showed decreased expression of PADI3 in biopsy samples of scalp skin obtained from patients with CCCA. We then directly sequenced PADI3 in an additional 42 patients (replication set) and observed genetic variants in 9 of them. A post hoc analysis of the combined data sets showed that the prevalence of PADI3 mutation was higher among patients with CCCA than in a control cohort of women of African ancestry (P = 0.002 by the chi-square test; P = 0.006 by Fisher's exact test; and after adjustment for relatedness of persons, P = 0.03 and P = 0.04, respectively). CONCLUSIONS: Mutations in PADI3, which encodes a protein that is essential to proper hair-shaft formation, were associated with CCCA. (Funded by the Ram Family Foundation and others.).

Impact of Post-Transplantation Hypomagnesemia on Long-Term Graft and Patient Survival after Transplantation.

BACKGROUND: Post-transplant hypomagnesemia is commonly observed among patients prescribed calcineurin inhibitor (CNIs). METHODS: We conducted a retrospective single-center analysis (2000-2013, N = 726) to examine the association of hypomagnesemia with long-term patient and allograft outcomes in kidney transplant recipients. A median serum magnesium (Mg) level of all measured Mg levels from 1 month to 1 year posttransplant was calculated. RESULTS: For every increase in Mg of 0.1 mg/dL, the risk for either graft loss or death, overall mortality, and death with a functioning graft increased by 11%, 14%, and 12%, respectively (p < 0.01). In a multivariate model, patients with median Mg level ≥1.7 mg/dL had a reduced overall survival rate (HR 1.57, 95% CI: 1.04-2.38, p = 0.033) compared to those with median Mg level <1.7 mg/dL. This association was observed in subgroups of patients above 60 years old, in those who had a slow graft function (SGF) and in females. CONCLUSIONS: Posttransplant hypomagnesemia is associated with better patient and allograft survival up to 10 years posttransplant. This relationship remained significant after accounting for baseline allograft function, presence of SGF and CNI trough levels.

Rapid 8-Zone Lung Ultrasound Protocol is Comparable to a Full 12-Zone Protocol for Outcome Prediction in Hospitalized COVID-19 Patients.

OBJECTIVES: Safety precautions limit the clinical assessment of hospitalized Coronavirus disease 2019 (COVID-19) patients. The minimal exposure required to perform lung ultrasound (LUS) paired with its high accuracy, reproducibility, and availability make it an attractive solution for initial assessment of COVID-19 patients. We aim to evaluate whether the association between sonographic findings and clinical outcomes among COVID 19 patients is comparable between the validated 12-zone protocol and a shorter, 8-zone protocol, in which the posterior lung regions are omitted. METHODS: One hundred and one COVID-19 patients hospitalized in a dedicated COVID-19 ward in a tertiary referral hospital were examined upon admission and scored by 2 LUS protocols. The association between the scores and a composite outcome consisting of death, transfer to the intensive care unit (ICU) or initiation of invasive or noninvasive mechanical ventilation was estimated and compared. RESULTS: LUS scores in both the 8- and the 12-zone protocols were associated with the composite outcome during hospitalization (hazard ratio [HR] 1.21 [1.03-1.42, P = .022] and HR 1.13 [1.01-1.27, P = .037], respectively). The observed difference in the discriminatory ROC-AUC values for the 8- and 12-zone scores was not significant (0.767 and 0.754 [P = .647], respectively). CONCLUSION: A short 8-zone LUS protocol is as accurate as the previously validated, 12-zone protocol for prognostication of clinical deterioration in nonventilated COVID-19 patients.

Targeted therapies for immune thrombocytopenic purpura: a meta-analysis of randomized controlled trials.

Several targeted therapies have been approved in recent years for second-line treatment of immune thrombocytopenic purpura (ITP), providing an alternative to rituximab and splenectomy. The extent to which these drugs reduce bleeding risk has not been well defined. Targeted therapies recently approved for the treatment of ITP in adults were identified through a search of recently published professional guidelines. Randomized controlled trials (RCTs) supporting regulatory approval were identified through a search of drug labels on FDA@gov. Odds ratios (ORs) and associated 95% confidence intervals (CIs) were computed for pre-specified efficacy outcomes including platelet recovery to ≥ 50,000/µL, major and minor bleeding events, and survival. ORs for all adverse events were also computed. Four targeted therapies were identified, including three thrombopoietin receptor agonists and one tyrosine kinase inhibitor. Six RCTs, comprising 752 patients, were included in the meta-analysis. More patients treated with targeted therapies for ITP as compared to placebo achieved platelet counts over ≥ 50,000/µL (OR 8.29, 95% CI 5.59-12.29). Compared to placebo, targeted therapies for ITP were associated with significantly lower odds for major bleeding (OR 0.43, 95% CI 0.21-0.91), minor bleeding (OR 0.66, 95% CI 0.45-0.97), and with numerically lower mortality rates (OR 0.24, 95% CI 0.05-1.07). The odds for adverse events were comparable between the two arms (OR 1.43 95% CI 0.76-2.67). Compared to placebo, targeted therapies for ITP increase platelet counts, decrease bleeding events, and show a trend towards lower mortality, without increased toxicity. These findings support their use as a second-line ITP treatment.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing.

OBJECTIVES: Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. METHODS: To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. RESULTS: Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. CONCLUSIONS: Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Efficacy of a second external cephalic version (ECV) after a successful first external cephalic version with subsequent spontaneous reinversion to breech presentation: a retrospective cohort study.

OBJECTIVE: Determining the efficacy of performance of a second external cephalic version (ECV) following successful first ECV with subsequent spontaneous reinversion to breech presentation in reducing the rate of cesarean delivery (CD). METHODS: Data were reviewed on healthy women with fetuses in breech presentation who underwent a first ECV after 36 weeks. Routine ultrasound study was performed at 39-week gestation, and a repeat ECV procedure was performed if the fetus had reverted to non-cephalic presentation. Obstetrical outcome measures were compared between women who underwent one successful ECV between 36- and 41-week gestation in which the fetus remained in cephalic presentation until labor and those who underwent a successful first ECV after which the fetus returned to breech and a second ECV was performed. The primary outcome was the rate of secondary CD during vaginal delivery in cephalic presentation; rate of successful second ECV was the secondary outcome. RESULTS: Overall 250 women underwent one ECV attempt of which 169 (67%) were successful. Of them 28 reverted to breech presentation, all women underwent two attempts of which 21 (76%) were successful. A second successful ECV attempt was associated with a 33% incidence of a CD vs. 2.8% after one successful ECV in which the fetus remained in cephalic presentation. CONCLUSION: A second ECV after a successful first ECV with subsequent spontaneous reversion to breech presentation can be expected to be successful in 76% of cases but lead to CD in 33% of cases. Our findings can be used to support patient counseling and decision-making before second ECV attempt.

Can uric acid blood levels in renal transplant recipients predict allograft outcome?

BACKGROUND: Hyperuricemia is common after renal transplantation, especially in those receiving calcineurin inhibitors. Little, however, is known about the relationship between uric acid (UA) levels and allograft outcome. METHODS: We conducted a retrospective single-center analysis (N = 368) in order to assess UA blood levels post-transplant association with allograft outcome. For this study, a median serum UA level of all measured UA levels from 1 month to 1 year post renal transplantation was calculated. RESULTS: Patients were divided into 2 groups based on the median UA level measured between 1 and 12 months post-transplant. Those with median UA level ≥ 7 and ≥ 6 mg/dL (N = 164) versus median UA level < 7 and < 6 mg/dL for men and women respectively (N = 204) had lower GFR values at 1, 3 and 5 years posttransplant (mean GFR ± SD of 43.4 ± 20.6 and 58 ± 19.9 at 3 years post-transplant, p < 0.001). In multivariate models, UA levels were no longer significantly associated with renal allograft function. In a multivariate cox proportional hazard model, UA level was found to be independently associated with increased risk for death-censored graft loss (HR of 1.3, 95% CI 1.0-1.7, p < 0.05 for every increase of 1 mg/dL in UA level). CONCLUSION: Hyperuricemia was found to be associated with increased death- censored graft loss but not with allograft function. Increased UA levels were not found to be an independent predictor of long-term allograft function despite the known association of hyperuricemia with the progression of cardiovascular and renal disease.

Spectrum of genes for inherited hearing loss in the Israeli Jewish population, including the novel human deafness gene ATOH1.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss: ATOH1 (Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results show that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

The effects of a postmiscarriage menstrual period prior to reconceiving.

BACKGROUND: The optimal interpregnancy interval after a single pregnancy loss is controversial. It is common obstetrical practice to recommend that women who have had a miscarriage in the first trimester of pregnancy should wait for 1 or more menstrual cycles before attempting to conceive again. OBJECTIVE: This study aimed to assess whether conception before the first menstrual period after a spontaneous pregnancy loss is associated with a risk of repeat miscarriage or adverse perinatal outcomes. STUDY DESIGN: This retrospective cohort study included 107 women who had a spontaneous miscarriage in the first trimester of pregnancy followed by a subsequent pregnancy with an interpregnancy interval of <12 weeks. All miscarriages had ended in either spontaneous expulsion of the products of conception or medical or surgical evacuation of the uterus. The perinatal outcome measures of 57 women who conceived after the first menstrual period following a spontaneous miscarriage were compared with perinatal outcome measures of 50 women who conceived before the first menstrual period following a spontaneous miscarriage. The primary outcome was rate of pregnancy loss, and the secondary outcomes were gestational age at delivery and birthweight. RESULTS: The rate of recurrent miscarriage was 10.4% for women who conceived before the first menstrual period following a spontaneous miscarriage and 15.8% for those who conceived after (P=.604). There were no differences in the gestational age at delivery (38.9 vs 38.7 weeks; P=.66) or the birthweight (3347±173 vs 3412±156 g; P=.5) between the 2 groups. Other outcomes, such as mode of delivery and 5-minute Apgar score, were also similar for both groups. A multiple logistic regression analysis confirmed that conception before the first menstrual period following a spontaneous miscarriage was not associated with a higher incidence of subsequent miscarriage (odds ratio, 1.74; P=.46) or any other untoward outcome. CONCLUSION: Conception shortly after a spontaneous miscarriage without waiting for at least the first postmiscarriage menstrual period is not associated with adverse maternal or neonatal outcomes compared with those of women with similar interpregnancy intervals who conceived after their next menstrual period.

Early persistent hyperparathyroidism post-renal transplantation as a predictor of worse graft function and mortality after transplantation.

BACKGROUND: Persistent hyperparathyroidism (pHPT) is frequently seen after transplantation contributing to post-transplant complications. METHODS: We conducted a retrospective single center analysis to explore the relationship of early pHPT and long-term allograft outcome. Patients were divided into high (N = 153) and low (N = 252) PTH groups based on serum parathyroid hormone (PTH) level 3 months post-transplant (PTH ≥ 150 and < 150 pg/mL, respectively). RESULTS: High PTH was found to be an independent predictor for reduced kidney allograft function up to 3 years post-transplant. eGFR decreased by 11.4 mL/min (P < .001) and the odds of having an eGFR < 60 mL/min 3 years post-transplant were sixfold higher (P < .01) in the high compared to the low PTH group. Subgroup analysis based on eGFR 1 year post-transplant, presence of slow graft function (SGF), and transplant type revealed similar results. High PTH three months post-transplant was also independently associated with an increased risk for overall mortality and for death with a functioning graft (P < .05). CONCLUSIONS: pHPT three months post-renal transplantation is an independent predictor for a worse allograft function up to 3 years post-transplant and a risk factor for mortality. This relationship remains statistically significant after accounting for baseline allograft function, presence of SGF and serum mineral levels abnormalities.

Posttransplantation Hypomagnesemia as a Predictor of Better Graft Function after Transplantation.

BACKGROUND: Hypomagnesemia is frequently seen after transplantation and is particularly associated with the use of calcineurin inhibitors (CNIs). METHODS: We conducted a retrospective, single-center analysis (2000-2013, N = 726) to explore the relationship between hypomagnesemia and long-term allograft outcome in kidney transplant recipients. For this study, a median serum magnesium (Mg) level of all measured Mg levels from 1 month to 1 year after renal transplantation was calculated. RESULTS: For every increase in Mg by 0.1 mg/dL, the GFR decreased by 1.1 mL/min at 3 years posttransplant (p < 0.01) and by 1.5 mL/min at 5 years posttransplant. A median blood Mg level of ≥1.7 was found to be an independent predictor of a GFR <60 mL/min at 3 years posttransplant. The odds of having a GFR <60 mL/min 3 years posttransplant was almost 2-fold higher in the high Mg group than in the low Mg group. CONCLUSIONS: Hypomagnesemia from 1 to 12 months after renal transplantation is associated with a better allograft function up to 5 years posttransplant. This relationship was found to hold true after accounting for baseline allograft function and the presence of slow graft function.

Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT).

Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.

MicroRNA-mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV-1 infection.

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV-1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV-1 inhibition mechanism. As a result, it is expected that during HIV-1 infection miRNAs target genes that hinder or prevent the progression of the HIV-1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV-1 infection are cellular restriction factors. In this study, we identified a direct miRNA target gene that modulates viral spread in T-lymphocytes and HeLa-CCR5 cell lines. Following infection, let-7c, miR-34a or miR-124a were upregulated, and they targeted and downregulated p21 and TASK1 (also known as CDKN1A and KCNK3, respectively) cellular proteins. This eventually led to increased virion release and higher copy number of viral genome transcripts in infected cells. Conversely, by downregulating these miRNAs, we could suppress viral replication and spread. Our data suggest that HIV-1 exploits the host miRNA cellular systems in order to block the innate inhibition mechanism, allowing a more efficient infection process.

Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype.

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.

Epidermolytic Ichthyosis Sine Epidermolysis.

Epidermolytic ichthyosis (EI) is a rare disorder of cornification caused by mutations in KRT1 and KRT10, encoding two suprabasal epidermal keratins. Because of the variable clinical features and severity of the disease, histopathology is often required to correctly direct the molecular analysis. EI is characterized by hyperkeratosis and vacuolar degeneration of the upper epidermis, also known as epidermolytic hyperkeratosis, hence the name of the disease. In the current report, the authors describe members of 2 families presenting with clinical features consistent with EI. The patients were shown to carry classical mutations in KRT1 or KRT10, but did not display epidermolytic changes on histology. These observations underscore the need to remain aware of the limitations of pathological features when considering a diagnosis of EI.

Cole Disease Results from Mutations in ENPP1.

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.

Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity.

UNLABELLED: Coronaviruses (CoVs) are unique in encoding a 3'→5' exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small nonenzymatic protein. However, it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine substitution mutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN(+)] at positions within nsp10 known to disrupt the nsp10-nsp14 interaction in vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(+), was five to ten times more sensitive to treatment with the RNA mutagen 5-fluorouracil (5-FU) than wild-type (WT)-ExoN(+), suggestive of decreased replication fidelity. This decreased-fidelity phenotype was confirmed using two additional nucleoside analogs, 5-azacytidine and ribavirin. R80A/E82A-ExoN(+) reached a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen with WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(-) relative to MHV-ExoN(-), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN(-) is linked to the presence of ExoN activity. Our results demonstrate that nsp10 is important for CoV replication fidelity and support the hypothesis that nsp10 functions to regulate nsp14-ExoN activity during virus replication. IMPORTANCE: The adaptive capacity of CoVs, as well as all other RNA viruses, is partially attributed to the presence of extensive population genetic diversity. However, decreased fidelity is detrimental to CoV replication and virulence; mutant CoVs with decreased replication fidelity are attenuated and more sensitive to inhibition by RNA mutagens. Thus, identifying the viral protein determinants of CoV fidelity is important for understanding CoV replication, pathogenesis, and virulence. In this report, we show that nsp10, a small, nonenzymatic viral protein, contributes to CoV replication fidelity. Our data support the hypothesis that CoVs have evolved multiple proteins, in addition to nsp14-ExoN, that are responsible for maintaining the integrity of the largest known RNA genomes.