Pathway Analysis using Gene-expression Profiles of HPV-positive and HPV-negative Oropharyngeal Cancer Patients in a Hispanic Population: Methodological Procedures.

OBJECTIVE: The incidence of oral cavity and pharyngeal cancer in Puerto Rican men is higher than it is in the men of any other ethnic/racial group in the United States of America (US). The information regarding the effect of the human papilloma virus (HPV) in the gene-expression profile among patients with this cancer is limited in Hispanic community. We aim to describe the methodology for future studies to identify the molecular networks for determining overrepresented signaling and metabolic canonical pathways, based on the differential gene-expression profiles of HPV+ and HPV- samples from patients with oropharyngeal squamous cell carcinoma in Puerto Rico. METHODS: We analyzed the RNA expression of 5 tissue samples from subjects diagnosed with oropharyngeal squamous cell carcinoma, 2 HPV+ and 3 HPV-, using Affymetrix GeneChips. The relative difference between the average gene expressions of the HPV+ and HPV- samples was assessed, based on the fold change (log2-scale). RESULTS: Our analysis revealed 10 up regulated molecules (Mup1, LRP1, P14KA, ALYREF, and BHMT) and 5 down regulated ones (PSME4, KEAP1, ELK3, FAM186B, and PRELID1), at a cutoff of 1.5-fold change. Ingenuity Pathway Analysis showed the following biological functions to be affected in the HPV+ samples: cancer, hematological disease, and RNA post-transcriptional modification. QRT-PCR analysis confirmed only the differential regulation of ALYREF, KEAP1, and FAM186B genes. CONCLUSION: The relevant methodological procedures described are sufficient to detect the most significant biological functions and pathways according to the HPV status in patients with oropharyngeal cancer in Puerto Rico.

After an initial Hermansky-Pudlak syndrome clinical diagnosis, molecular testing reveals variants for oculocutaneous albinism type 1B: A case report.

BACKGROUND: Albinism is a heterogeneous condition in which patients present complete absence, reduction, or normal pigmentation in skin, hair and eyes in addition to ocular defects. One of the heterogeneous forms of albinism is observed in Hermansky-Pudlak syndrome (HPS) patients. HPS is characterized by albinism and hemorrhagic diathesis due to the absence of dense bodies in platelets. METHODS: In this report, we describe a case of a pair of Puerto Rican siblings with albinism that were clinically diagnosed with HPS during childhood. Since they did not harbor the founder changes in the HPS1 and HPS3 genes common in Puerto Ricans, as adults they wanted to know the type of albinism they had. We performed exome sequencing, validation by PCR, and cloning of PCR products followed by Sanger sequencing in the family members. RESULTS: We discovered no mutations that could explain an HPS diagnosis. Instead, we found the siblings were compound heterozygotes for 4 variants in the Tyrosinase gene: c.-301C>T, c.140G>A (rs61753180; p.G47D), c.575C>A (rs1042602; p.S192Y), and c.1205G>A (rs1126809; p.R402Q). Our results show that the correct diagnosis for the siblings is OCA1B. CONCLUSION: Our study shows the importance of molecular testing when diagnosing a rare genetic disorder, especially in populations were the disease prevalence is higher.

Molecular profiling of colorectal cancer in a genetically admixed Hispanic population.

BACKGORUND: Colorectal cancer (CRC) is among the leading causes of cancer-related deaths among Hispanics living in the United States (USH). Understanding the most common carcinogenic molecular pathways that affect Hispanics with CRC is crucial to guide research efforts in developing new therapeutic modalities incorporating genomically diverse populations. Tumor profiling techniques help identify actionable alternatives to recommend treatment and improve survival in cancer patients. METHODS: We conducted a secondary data analysis to evaluate the mutational profile of 218 CRC tumors in Hispanics living in Puerto Rico (PRH) who underwent next-generation sequencing (NGS) testing from 2015 to 2020. We compared the prevalence of CRC tumor somatic mutations in PRHs with the mutational profiles reported for CRC from The Cancer Genome Atlas (TCGA) Pan-Cancer Clinical Data, the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE)-Non-Hispanic, and GENIE-Hispanic datasets. RESULTS: Among the top mutated genes in CRC tumors in PRHs were APC, TP53, and KRAS, which had significantly higher mutational frequencies in PRH compared to the examined datasets, including GENIE-Hispanics. The most frequent gene amplifications for PRH were CDX2, CDKN1B, and HNRNPA2B1. Targetable biomarkers for CRC, such as microsatellite instability-high (MSI), wild-type KRAS, wild-type NRAS, V600E BRAF, and ERBB2 gene amplifications were found in 2.0%, 43.8%, 97.8%, 3.9%, and 2.3%, respectively, of PRH patients. CONCLUSION: This is the first study to report the mutational profile of CRC tumors in PRHs and make comparisons to other non-Hispanic and USH populations.

Incidence and Mortality Rates for Childhood Acute Lymphoblastic Leukemia in Puerto Rican Hispanics, 2012-2016.

BACKGROUND: Acute lymphoblastic leukemia (ALL) accounts for 80% of all leukemias diagnosed in children. Although ALL age patterns are consistent across racial/ethnic groups, their incidence and mortality rates are highly variable. We assessed the age-standardized ALL incidence and mortality rates of Puerto Rican Hispanic (PRH) children and compared them with those of US mainland Hispanics (USH), non-Hispanic Whites (NHW), non-Hispanic Blacks (NHB), and Non-Hispanic Asian or Pacific Islanders (NHAPI). METHODS: Differences between racial/ethnic groups were assessed by estimating the standardized rate ratio (SRR) for 2010 to 2014. Secondary data analyses of the Puerto Rico Central Cancer Registry and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) databases were performed for the 2001 to 2016 period. RESULTS: PRH children had 31% lower incidence rates than USH, but 86% higher incidence rates than NHB. In addition, the incidence trends of ALL increased significantly from 2001 to 2016 among PRH and USH, with 5% and 0.9% per year, respectively. Moreover, PRH have a lower 5-year overall survival (81.7%) when compared with other racial/ethnic groups. CONCLUSIONS: PRH children were found to have disparities in ALL incidence and mortality rates compared with other racial/ethnic groups in the US. Additional research is warranted to identify the genetic and environmental risk factors that may be associated with the disparities observed. IMPACT: This is the first study reporting the incidence and mortality rates of childhood ALL for PRH and making comparisons with other racial/ethnic groups in the US. See related commentary by Mejía-Aranguré and Núñez-Enríquez, p. 999.

Molecular and Sociodemographic Colorectal Cancer Disparities in Latinos Living in Puerto Rico.

BACKGROUND: The incidence of sporadic colorectal cancer (CRC) among individuals <50 years (early-onset CRC) has been increasing in the United States (U.S.) and Puerto Rico. CRC is currently the leading cause of cancer death among Hispanic men and women living in Puerto Rico (PRH). The objective of this study was to characterize the molecular markers and clinicopathologic features of colorectal tumors from PRH to better understand the molecular pathways leading to CRC in this Hispanic subpopulation. METHODS: Microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and KRAS and BRAF mutation status were analyzed. Sociodemographic and clinicopathological characteristics were evaluated using Chi-squared and Fisher's exact tests. RESULTS: Of the 718 tumors analyzed, 34.2% (n = 245) were early-onset CRC, and 51.7% were males. Among the tumors with molecular data available (n = 192), 3.2% had MSI, 9.7% had BRAF, and 31.9% had KRAS mutations. The most common KRAS mutations observed were G12D (26.6%) and G13D (20.0%); G12C was present in 4.4% of tumors. A higher percentage of Amerindian admixture was significantly associated with early-onset CRC. CONCLUSIONS: The differences observed in the prevalence of the molecular markers among PRH tumors compared to other racial/ethnic groups suggest a distinct molecular carcinogenic pathway among Hispanics. Additional studies are warranted.

Bioinformatic Characterization and Molecular Evolution of the Lucina pectinata Hemoglobins.

(1) Introduction: Lucina pectinata is a clam found in sulfide-rich mud environments that has three hemoglobins believed to be responsible for the transport of hydrogen sulfide (HbILp) and oxygen (HbIILp and HbIIILp) to chemoautotrophic endosymbionts. The physiological roles and evolution of these globins in sulfide-rich environments are not well understood. (2) Methods: We performed bioinformatic and phylogenetic analyses with 32 homologous mollusk globin sequences. Phylogenetics suggests a first gene duplication resulting in sulfide binding and oxygen binding genes. A more recent gene duplication gave rise to the two oxygen-binding hemoglobins. Multidimensional scaling analysis of the sequence space shows evolutionary drift of HbIILp and HbIIILp, while HbILp was closer to the Calyptogena hemoglobins. Further corroboration is seen by conservation in the coding region of hemoglobins from L. pectinata compared to those from Calyptogena. (3) Conclusions: Presence of glutamine in position E7 in organisms living in sulfide-rich environments can be considered an adaptation to prevent loss of protein function. In HbILp a substitution of phenylalanine in position B10 is accountable for its unique reactivity towards H2S. It appears that HbILp has been changing over time, apparently not subject to functional constraints of binding oxygen, and acquired a unique function for a specialized environment.

Toward the discovery of biological functions associated with the mechanosensor Mtl1p of Saccharomyces cerevisiae via integrative multi-OMICs analysis.

Functional analysis of the Mtl1 protein in Saccharomyces cerevisiae has revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests that mtl1Δ strains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed that mtl1Δ strains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased expression of anabolic enzymes and increased expression of catabolic enzymes involved in the metabolism of amino acids, with enhanced expression of mitochondrial respirasome proteins. These observations support the idea that Mtl1 protein controls the suppression of a non-lethal heat stress response under normal conditions while it plays an important role in metabolic regulatory mechanisms linked to TORC1 signaling that are required to maintain cellular homeostasis and optimal mitochondrial function.

Characterization of Histone Genes from the Bivalve Lucina Pectinata.

Lucina pectinata is a clam that lives in sulfide-rich environments and houses intracellular sulfide-oxidizing endosymbionts. To identify new Lucina pectinata proteins, we produced libraries for genome and transcriptome sequencing and assembled them de novo. We searched for histone-like sequences using the Lucina pectinata histone H3 partial nucleotide sequence against our previously described genome assembly to obtain the complete coding region and identify H3 coding sequences from mollusk sequences in Genbank. Solen marginatus histone nucleotide sequences were used as query sequences using the genome and transcriptome assemblies to identify the Lucina pectinata H1, H2A, H2B and H4 genes and mRNAs and obtained the complete coding regions of the five histone genes by RT-PCR combined with automated Sanger DNA sequencing. The amino acid sequence conservation between the Lucina pectinata and Solen marginatus histones was: 77%, 93%, 83%, 96% and 97% for H1, H2A, H2B, H3 and H4, respectively. As expected, the H3 and H4 proteins were the most conserved and the H1 proteins were most similar to H1's from aquatic organisms like Crassostrea gigas, Aplysia californica, Mytilus trossulus and Biomphalaria glabrata. The Lucina pectinata draft genome and transcriptome assemblies, obtained by semiconductor sequencing, were adequate for identification of conserved proteins as evidenced by our results for the histone genes.

Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes.

The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5'end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments.