Genomic analysis of a Palestinian family with inherited cancer syndrome: a next-generation sequencing study.

Familial predisposition is a strong risk factor for different types of cancer and accounts for around 10% of the cases. In this study, we investigated cancer predisposition in a Palestinian family using whole-exome sequencing (WES) technologies. In this study, we focused more on cutaneous melanoma (CM). Our analysis identified three heterozygous rare missense variants, WRN (p.L383F and p.A995T) and TYRP1 (p.T262M) and a pathogenic homozygous missense mutation in ERCC2 (p.R683Q). Although WRN and TYRP1 genes and their variations were correlated with different types of cancer, including melanoma, the currently identified WRN and TYRP1 variants were not reported previously in melanoma cases. The pathogenic mutation was segregated with the clinical phenotypes and found in the two affected brothers, one with CM and the other with brain tumor, and was confirmed by Sanger sequencing analysis. Segregation analysis of this mutation revealed that family members are either heterozygous or wild type. Our findings confirm that the homozygous ERCC2 (p.R683Q) mutation was responsible for causing melanoma and other cancer types in the family. Our work highlights the value to decipher the mutational background of familial cancers, especially CM, in the Palestinian population to guide diagnosis, prevention, and treatment of affected patients and their families.

Signatures of historical demography and pathogen richness on MHC class I genes.

The extreme polymorphism of MHC class I has been argued to be driven by balancing selection from pathogens, with the prediction that populations exposed to a wider variety of diseases should have higher diversity. We assembled a global database of allotype frequencies for MHC class I genes and investigated possible drivers of genetic diversity, measured in different ways. We first looked for a decline in diversity with distance from Africa (a consequence of drift during human expansions) and then investigated the link with pathogen richness once the effect of drift had been corrected for. Using heterozygosity, we recovered a clear decline in diversity from Africa and confirmed the positive relationship between genetic diversity and pathogen richness for all three classical MHC class I genes. However, when we considered a sequence-based measure of genetic diversity, the correlation with geographic distance from Africa vanished for HLA-C, and the correlations with pathogen richness for the three MHC class I genes were much weaker. HLA-C is known to consist of two functional classes of allotypes (classified with respect to the 80th residue), which interact with different KIR receptors. While this separation provided some improvement in the fit between genetic diversity and distance from Africa for one class, much clearer and consistent patterns were recovered when we used the 90th residue to separate HLA-C allotypes into two new classes. This suggests that this residue, which is also involved in the binding of KIR, might have had an important evolutionary role that has been overlooked.

Seroprevalence of SARS-CoV-2 Antibodies among Vaccinated and Non-Vaccinated Adults in the West Bank: Results of a Repeated Cross-Sectional Study.

Seroprevalence studies provide an accurate measure of SARS-CoV-2 spread at a population level and the number of undiagnosed individuals. Repeated cross-sectional sero-studies are encouraged to monitor the spread of the virus. The aim of this study is to assess the seroprevalence rate among a random sample of Palestinians residing in the West Bank region of Palestine, especially among those who were not vaccinated and not diagnosed. The study was able to assess the prevalence of asymptomatic cases among the Palestinian adult population. The study also focused on measuring the percentage of adult Palestinians who accepted to get vaccinated across gender and age groups. Methods: This second round cross-sectional study involved 1451 participants, who agreed to be interviewed and answer the questionnaire, where 910 of them agreed to participate in the sero-study and donate a blood sample to be tested for antibodies. The sample was randomly selected from the adult population, 18 years or older, living in the West Bank region of Palestine. Serological tests for 910 adequate serum samples were performed using immunoassays for the detection of antibodies against SARS-CoV-2. Sociodemographic information and medical history data were collected. Results: Study findings indicate that as of October 2021, there was a seroprevalence rate of 75.9% (30% due to infection with COVID-19 virus and 45.9% due to vaccination) with 95% CI (73.1−78.7). The results indicate that the prevalence of antibodies among those who are unvaccinated and undiagnosed was 45.2% with 95% CI (39.9−50.5%). The average age of participants was 37.6 years old. A total of 49.2% were females, and 50.8% were males. In relation to COVID-19, 13.6% of respondents reported getting infected by COVID-19 with statistically significant difference (p-value = 0.001) between males (10.7%) and females (16.5%). In terms of vaccination, 52.8% of respondents reported getting vaccinated with an important difference between males (64.3%) and females (40.9%), (p-value < 0.01). Conclusions: Our findings reveal a drastic rise in seroprevalence of SARS-CoV-2 antibodies due to infection and vaccination. This information is useful for assessing the degree of herd immunity among the adult population and provides better understanding of the pandemic. Population-based seroprevalence studies should be conducted periodically to monitor the SARS-CoV-2 seroprevalence in Palestine and inform policy makers about the efficacy of the surveillance system and the public compliance with vaccination policies especially among females.

Knowledge, attitudes and practices (KAP) towards COVID-19 among Palestinians during the COVID-19 outbreak: A cross-sectional survey.

Coronavirus disease 2019 (COVID-19) is a highly contagious illness that spreads rapidly through human-to-human transmission. On March 5, the government of Palestine declared a state of emergency in order to curb the spread of the virus, a declaration that it extended for a fifth time on July 5th. The degree to which a population complies with corresponding safety measures is surely affected by the people's knowledge, attitudes and practices (KAP) towards the disease. To explore this hypothesis, we gathered data from 1,731 Palestinians between April 19thand May 1st, 2020 through a KAP questionnaire. The participant pool represented a stratified sample of Palestinians living across a number of governorates in the Gaza Strip and the West Bank, with 36.5% from Gaza and (63.5%) from the West Bank. Gender was almost equally distributed within the sample with (51%) men respondents and (49%) women respondent. The questionnaire included 17 questions about participants' knowledge and awareness of COVID-19, 17 questions regarding the safety measures they had taken in the wake of the outbreak and 3 questions asking them to assess the efficacy of the government's response to the pandemic. Our data shows that 79% of the respondents have good awareness about transmission of the virus, 55.6% were knowledgeable of the symptoms exhibited by an infected individual, 81% were aware of the preventative measures and 82% demonstrated awareness of the risk groups. Most participants complied with preventative measures (77%) and 62% the study participants agreed that stricter measures have to be enforced by the government to limit the spread of the virus. Our study revealed that younger participants and people with higher educational level demonstrated more awareness of the virus. Also, Women were reported to be more aware of preventative measures and to have complied more with good practices. We report that residents of the West Bank have complied more with the right practices when compared to residents of Gaza. Based on the results of this study, we conclude that health education programs aimed at improving the public's understanding of COVID-19 are important in helping the population maintain appropriate practices and should be target people with lower educational level, and that findings such as those discussed in this report may provide valuable feedback to lawmakers working to stop the spread of the virus.

Seroprevalence of SARS-CoV-2 in the West Bank region of Palestine: a cross-sectional seroepidemiological study.

OBJECTIVES: Seroprevalence rates are important indicators to the epidemiology of COVID-19 and the extent of the pandemic given the existence of asymptomatic cases. The purpose of this study is to assess the seroprevalence rate in the Palestinian population residing in the West Bank. SETTING: The study involved 1355 participants from 11 governorates, including 112 localities in the West Bank and 1136 individuals visiting Palestinian medical laboratories. PARTICIPANTS: Blood samples were collected between 15th June 2020 and 30th June 2020 from 1355 individuals from randomly selected households in the West Bank, in addition to 1136 individuals visiting Palestinian medical laboratories between the 1st May 2020 and 9th July 2020 for a routine check-up. PRIMARY AND SECONDARY OUTCOME MEASURES: Out of the 2491 blood samples collected, serological tests for 2455 adequate serum samples were done using an immunoassay for qualitative detection of antibodies against SARS-CoV-2. Seroprevalence was estimated as the proportion of individuals who had a positive result in the total SARS-CoV-2 antibodies in the immunoassay. RESULTS: The random sample of Palestinians living in the West Bank yielded 0% seroprevalence with 95% and an adjusted CI (0% to 0.0043%), while the lab referral samples yielded an estimated seroprevalence of 0.354% with 95% and an adjusted CI (0.001325% to 0.011566%). CONCLUSIONS: Our results indicate that as of mid-June 2020, seroprevalence in Palestine persists low and is inadequate to provide herd immunity, emphasising the need to maintain health measures to keep the outbreak under control. Population-based seroprevalence studies are to be conducted periodically to monitor the SARS-CoV-2 seroprevalence in Palestine and inform policymakers about the efficacy of their surveillance system.

Genomic epidemiology of the first epidemic wave of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Palestine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the COVID-19 pandemic, continues to cause a significant public-health burden and disruption globally. Genomic epidemiology approaches point to most countries in the world having experienced many independent introductions of SARS-CoV-2 during the early stages of the pandemic. However, this situation may change with local lockdown policies and restrictions on travel, leading to the emergence of more geographically structured viral populations and lineages transmitting locally. Here, we report the first SARS-CoV-2 genomes from Palestine sampled from early March 2020, when the first cases were observed, through to August of 2020. SARS-CoV-2 genomes from Palestine fall across the diversity of the global phylogeny, consistent with at least nine independent introductions into the region. We identify one locally predominant lineage in circulation represented by 50 Palestinian SARS-CoV-2, grouping with genomes generated from Israel and the UK. We estimate the age of introduction of this lineage to 05/02/2020 (16/01/2020-19/02/2020), suggesting SARS-CoV-2 was already in circulation in Palestine predating its first detection in Bethlehem in early March. Our work highlights the value of ongoing genomic surveillance and monitoring to reconstruct the epidemiology of COVID-19 at both local and global scales.

Consolidating biallelic SDHD variants as a cause of mitochondrial complex II deficiency.

Isolated mitochondrial complex II deficiency is a rare cause of mitochondrial respiratory chain disease. To date biallelic variants in three genes encoding mitochondrial complex II molecular components have been unequivocally associated with mitochondrial disease (SDHA/SDHB/SDHAF1). Additionally, variants in one further complex II component (SDHD) have been identified as a candidate cause of isolated mitochondrial complex II deficiency in just two unrelated affected individuals with clinical features consistent with mitochondrial disease, including progressive encephalomyopathy and lethal infantile cardiomyopathy. We present clinical and genomic investigations in four individuals from an extended Palestinian family with clinical features consistent with an autosomal recessive mitochondrial complex II deficiency, in which our genomic studies identified a homozygous NM_003002.3:c.[205 G > A];[205 G > A];p.[(Glu69Lys)];[(Glu69Lys)] SDHD variant as the likely cause. Reviewing previously published cases, these findings consolidate disruption of SDHD function as a cause of mitochondrial complex II deficiency and further define the phenotypic spectrum associated with SDHD gene variants.

Exome Sequencing of ABCB5 Identifies Recurrent Melanoma Mutations that Result in Increased Proliferative and Invasive Capacities.

ABCB5 is an ABC transporter that was shown to confer low-level multidrug resistance in cancer. In this study, we show that ABCB5 was mutated in 13.75% of the 640 melanoma samples analyzed. Besides nonsense mutations, two mutation hotspots were found in the ABCB5 protein, in the drug-binding pocket and the nucleotide-binding domains. Four mutations, which are representative of the mutation pattern, were selected. ATPase assays showed that these mutations resulted in a decrease in basal ATP hydrolysis by ABCB5. To select informative melanoma cell lines, mutational profiles of the clinical samples were further analyzed. This study showed mutations in the tumor suppressor CDKN2A gene and the NRAS oncogene in 62.5% and 75%, respectively of the samples that had mutations in the ABCB5 gene. No mutation was found in the tumor suppressor PTEN gene, whereas the activating V600E mutation in the BRAF oncogene was found in 25% of the samples with a mutated ABCB5 gene. Studies in four melanoma cell lines with various genetic backgrounds showed an increase in the proliferation and migration capacity of mutant ABCB5-expressing cells, suggesting that ABCB5 plays a role in the development of melanoma as a tumor suppressor gene.

Author Correction: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma.

The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor-intensive. To this end, we present a polyadenylation signal-trap construct for N'-tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c-CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.

Combined Analysis of Antigen Presentation and T-cell Recognition Reveals Restricted Immune Responses in Melanoma.

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma.

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.

RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells.

Analysis of 501 melanoma exomes revealed RGS7, which encodes a GTPase-accelerating protein (GAP), to be a tumor-suppressor gene. RGS7 was mutated in 11% of melanomas and was found to harbor three recurrent mutations (p.R44C, p.E383K and p.R416Q). Structural modeling of the most common recurrent mutation of the three (p.R44C) predicted that it destabilizes the protein due to the loss of an H-bond and salt bridge network between the mutated position and the serine and aspartic acid residues at positions 58 as 61, respectively. We experimentally confirmed this prediction showing that the p.R44C mutant protein is indeed destabilized. We further show RGS7 p.R44C has weaker catalytic activity for its substrate Gαo, thus providing a dual mechanism for its loss of function. Both of these effects are expected to contribute to loss of function of RGS7 resulting in increased anchorage-independent growth, migration and invasion of melanoma cells. By mutating position 56 in the R44C mutant from valine to cysteine, thereby enabling the formation of a disulfide bridge between the two mutated positions, we slightly increased the catalytic activity and reinstated protein stability, leading to the rescue of RGS7's function as a tumor suppressor. Our findings identify RGS7 as a novel melanoma driver and point to the clinical relevance of using strategies to stabilize the protein and, thereby, restore its function.

Parkinson disease (PARK) genes are somatically mutated in cutaneous melanoma.

OBJECTIVE: To assess whether Parkinson disease (PD) genes are somatically mutated in cutaneous melanoma (CM) tissue, because CM occurs in patients with PD at higher rates than in the general population and PD is more common than expected in CM cohorts. METHODS: We cross-referenced somatic mutations in metastatic CM detected by whole-exome sequencing with the 15 known PD (PARK) genes. We computed the empirical distribution of the sum of mutations in each gene (Smut) and of the number of tissue samples in which a given gene was mutated at least once (SSampl) for each of the analyzable genes, determined the 90th and 95th percentiles of the empirical distributions of these sums, and verified the location of PARK genes in these distributions. Identical analyses were applied to adenocarcinoma of lung (ADENOCA-LUNG) and squamous cell carcinoma of lung (SQUAMCA-LUNG). We also analyzed the distribution of the number of mutated PARK genes in CM samples vs the 2 lung cancers. RESULTS: Somatic CM mutation analysis (n = 246) detected 315,914 mutations in 18,758 genes. Somatic CM mutations were found in 14 of 15 PARK genes. Forty-eight percent of CM samples carried ≥1 PARK mutation and 25% carried multiple PARK mutations. PARK8 mutations occurred above the 95th percentile of the empirical distribution for SMut and SSampl. Significantly more CM samples harbored multiple PARK gene mutations compared with SQUAMCA-LUNG (p = 0.0026) and with ADENOCA-LUNG (p < 0.0001). CONCLUSIONS: The overrepresentation of somatic PARK mutations in CM suggests shared dysregulated pathways for CM and PD.

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.

The functional relevance of somatic synonymous mutations in melanoma and other cancers.

Recent technological advances in sequencing have flooded the field of cancer research with knowledge about somatic mutations for many different cancer types. Most cancer genomics studies focus on mutations that alter the amino acid sequence, ignoring the potential impact of synonymous mutations. However, accumulating experimental evidence has demonstrated clear consequences for gene function, leading to a widespread recognition of the functional role of synonymous mutations and their causal connection to various diseases. Here, we review the evidence supporting the direct impact of synonymous mutations on gene function via gene splicing; mRNA stability, folding, and translation; protein folding; and miRNA-based regulation of expression. These results highlight the functional contribution of synonymous mutations to oncogenesis and the need to further investigate their detection and prioritization for experimental assessment.

Recurrent inactivating RASA2 mutations in melanoma.

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

Somatic mutation of GRIN2A in malignant melanoma results in loss of tumor suppressor activity via aberrant NMDAR complex formation.

The ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDARs)) are composed of large complexes of multi-protein subunits creating ion channels in the cell plasma membranes that allow for influx or efflux of mono- or divalent cations (e.g., Ca(2+)) important for synaptic transmissions, cellular migration, and survival. Recently, we discovered the high prevalence of somatic mutations within one of the ionotropic glutamate receptors, GRIN2A, in malignant melanoma. Functional characterization of a subset of GRIN2A mutants demonstrated a loss of NMDAR complex formation between GRIN1 and GRIN2A, increased anchorage-independent growth in soft agar, and increased migration. Somatic mutation of GRIN2A results in a dominant negative effect inhibiting the tumor-suppressive phenotype of wild-type (WT) GRIN2A in melanoma. Depletion of endogenous GRIN2A in melanoma cells expressing WT GRIN2A resulted in increased proliferation compared with control. In contrast, short-hairpin RNA depletion of GRIN2A in mutant cell lines slightly reduced proliferation. Our data show that somatic mutation of GRIN2A results in increased survival, and we demonstrate the functional importance of GRIN2A mutations in melanoma and the significance that ionotropic glutamate receptor signaling has in malignant melanoma.

A highly recurrent RPS27 5'UTR mutation in melanoma.

The incidence of melanoma continues to rise globally and is increasing at a rate greater than any other cancer. To systematically search for new genes involved in melanomagenesis, we collated exome sequencing data from independent melanoma cohort datasets, including those in the public domain. We identified recurrent mutations that may drive melanoma growth, survival or metastasis, and which may hold promise for the design of novel therapies to treat melanoma. These included a frequent recurrent (i.e. hotspot) mutation in the 5' untranslated region of RPS27 in ~10% of samples. We show that the mutation expands the 5'TOP element, a motif known to regulate the expression of most of the ribosomal protein family, to which RPS27 belongs, and thus might sensitize the mutated transcript to growth-mediated regulation. This finding highlights not only the important role of non-protein coding genetic aberrations in cancer development but also their potential as novel therapeutic targets.