Susana Seixas

Allele-specific CDH1 downregulation and hereditary diffuse gastric cancer

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer susceptibility syndrome characterized by early-onset diffuse gastric cancer (DGC) and lobular breast cancer. E-cadherin (CDH1) heterozygous germline mutations and deletions are found in 40% of families. Independent of CDH1 alterations, most HDGC tumours display mislocalized or absent E-cadherin immunoexpression, therefore undetected defects at the CDH1 locus may still be involved. We aimed at determining whether CDH1 mutation-negative probands display germline CDH1 allele-specific expression (ASE) imbalance, using a single-nucleotide primer extension-based procedure and tried to uncover the underlying molecular defect. CDH1 ASE analysis was performed using three intragenic SNPs in RNA extracted from the blood of 21 cancer-free individuals and 22 HDGC probands (5 CDH1 mutation carriers and 17 CDH1 negative). Germline promoter methylation, deletions and haplotype-related susceptibility at the CDH1 locus were analysed. Both CDH1 alleles from cancer-free individuals displayed equivalent expression levels, whereas monoallelic CDH1 expression or high allelic expression imbalance (AI) was present in 80% of CDH1 mutant and 70.6% (n = 12) of CDH1-negative HDGC probands. Germline deletions and promoter hypermethylation were found in 25% of probands displaying high CDH1 AI. No particular haplotype was found to be associated with CDH1 high AI. Germline CDH1 AI is highly frequent among CDH1 mutation-negative probands but was not seen in cancer-free individuals. This implicates the CDH1 locus in the majority of mutation-negative HDGC families.

A model to infer the pathogenic significance of CDH1 germline missense variants

Germline mutations of the E-cadherin gene (CDH1) are involved in the tumorigenesis of hereditary diffuse gastric cancer (HDGC). Recent studies have highlighted the lifesaving potential of total prophylactic gastrectomy for CDH1 germline mutation carriers. In this regard, CDH1 germline mutations of the missense type represent a clinical burden in genetic counseling, as their pathogenic relevance is not straightforward. In this work, we have outlined a possible multivariate approach to infer the significance of such variants. We reviewed all HDGC-associated E-cadherin germline missense mutations reported to date. The information collected included: co-segregation of the mutation within pedigrees, frequency in healthy population control, recurrence in independent families, and functional in vitro and in silico data. We used the neighbor-joining method to group mutations according to the collected information and assessed the robustness of mutation clusters with a bootstrap test. CDH1 germline missense variants were classified according to the parameters defined in the multivariate analysis. This analysis allowed the distribution of the variants into two distinct groups: neutral variants vs mutations. The model described in this study provides an important tool that can ultimately improve the genetic counseling offered to the carriers of the germline CDH1 missense variants.

The Peopling of São Tomé (Gulf of Guinea): Origins of Slave Settlers and Admixture with the Portuguese

The geographic origins of African slave settlers and the Portuguese genetic contribution to the population of São Tomé (Gulf of Guinea) were assessed through the analysis of b-globin haplotypes in 44 chromosomes bearing the bS allele and through the study of the genetic variation in eight autosomal markers (APOA1, AT3, FY, LPL, OCA2, RB1, Sb19.3, and GC) informative for admixture in a sample of 224 individuals. The observed bS haplotype distribution (36.4% Bantu, 52.3% Benin, 4.5% Cameroon, 4.5% Senegal, and 2.3% atypical) is in accordance with the historical information on the major geographic sources of slave settlers of São Tomé, although it captures a more important contribution of Central-West Africa regions than previously anticipated. European admixture, estimated to be 10.7 ± 0.9%, has created a considerable level of genetic structure, as indicated by the finding of significant linkage disequilibrium between 33% of unlinked marker loci pairs. Recent admixture was found to have an important contribution to these values, since removal of individuals with Portuguese or Cape Verdian parents or grandparents from the sample dropped the miscegenation level to 6.5 ± 0.8% and reduced significant linkage disequilibrium to 11% of unlinked marker pairs. Taken together, these results indicate that the peopling of São Tomé might have provided one of the first examples of the combination of diverse African contributions and European admixture that emerged from the overseas population relocations promoted by the Atlantic slave trade.

Lobar Brain Hemorrhages and White Matter Changes: Clinical, Radiological and Laboratorial Profiles

Background: White matter changes have several histopathologic correlates including cerebral amyloid angiopathy (CAA). The aim of this study was to characterize the clinical, laboratorial and neuroradiological profile of a CAA-related lobar hemorrhages case series. Methods: A cohort of 50 consecutive patients with cerebral lobar hemorrhages was studied and clinical, radiological data and ApoE polymorphisms were analyzed. White matter changes were graded and microbleeds were characterized according to number and location using T2* MRI. Results: A statistically significant association was found between the prestroke cognitive performance and poststroke dementia and between hemorrhage volume and mortality. More severe white matter changes were found in probable CAA when comparing to possible CAA. The most prominent white matter lesions are associated with the presence and the number of microbleeds. The frequency of APOE υ2 and υ4 alleles was higher in this cohort when compared to a Northern Portuguese population. Conclusion: White matter changes are frequent in lobar hemorrhage patients and are associated with cortical microbleeds, the radiological hallmark of CAA. Therefore, white matter changes may be the sole phenotype of CAA and, potentially, involved in the pre-stroke cognitive impairment presented by the patients, which are genetically distinct from the population in general.

Influence of Apolipoprotein E Polymorphism on Cardiovascular Risk Factors in Obese Children

Aim: The main objective of the study was to determine whether risk factors associated with obesity are influenced by genetic variation of apolipoprotein E (ApoE). Methods: 81 obese children (mean age 9.4 ± 2.8 years) and an age-matched control group were included. Body composition, lipid profile, and glucose and insulin levels were evaluated according to international recommendations, and the blood pressure was measured by an oscillometric method. Results: The calculated frequencies of the ApoE alleles *2, *3, and *4 (0.04, 0.88 and 0.08) in obese children were similar to those of eutrophic age-matched controls (0.07, 0.82, and 0.11) and fitted the range of variation generally observed in southern European populations. Age, anthropometric parameters, body fat mass, and blood pressure were similar in E2/3, E3/3 and E4/3 genotypes. Total/high-density lipoprotein cholesterol and low-density lipoprotein/high-density lipoprotein cholesterol ratios were higher in the E4/3 group as compared with E3/3 (p < 0.01) and E2/3 (p < 0.05) groups. No differences concerning clusters of risk factors were observed among the three genotypes. No associations were found between ApoE polymorphism and glucose levels (fasting and at 2 h) and between fasting insulin levels and HOMAIR results. Higher levels of fasting and 2-hour insulin and higher HOMAIR values were significantly associated with a higher fat mass. Conclusions: ApoE polymorphism seems to influence some lipid profile abnormalities associated with obesity in childhood. However, clustering of risk factors and insulin resistance seem not to be dependent on ApoE polymorphism.

Severe α‐1 antitrypsin deficiency caused by Q0Ourém allele: clinical features, haplotype characterization and history

Vaz Rodrigues L, Costa F, Marques P, Mendonça C, Rocha J, Seixas S. Severe α‐1 antitrypsin deficiency caused by Q0Ourém allele: clinical features, haplotype characterization and history.

Reproduction and Immunity-Driven Natural Selection in the Human WFDC Locus

The whey acidic protein (WAP) four-disulfide core domain (WFDC) locus located on human chromosome 20q13 spans 19 genes with WAP and/or Kunitz domains. These genes participate in antimicrobial, immune, and tissue homoeostasis activities. Neighboring SEMG genes encode seminal proteins Semenogelin 1 and 2 (SEMG1 and SEMG2). WFDC and SEMG genes have a strikingly high rate of amino acid replacement (dN/dS), indicative of responses to adaptive pressures during vertebrate evolution. To better understand the selection pressures acting on WFDC genes in human populations, we resequenced 18 genes and 54 noncoding segments in 71 European (CEU), African (YRI), and Asian (CHB + JPT) individuals. Overall, we identified 484 single-nucleotide polymorphisms (SNPs), including 65 coding variants (of which 49 are nonsynonymous differences). Using classic neutrality tests, we confirmed the signature of short-term balancing selection on WFDC8 in Europeans and a signature of positive selection spanning genes PI3, SEMG1, SEMG2, and SLPI. Associated with the latter signal, we identified an unusually homogeneous-derived 100-kb haplotype with a frequency of 88% in Asian populations. A putative candidate variant targeted by selection is Thr56Ser in SEMG1, which may alter the proteolytic profile of SEMG1 and antimicrobial activities of semen. All the well-characterized genes residing in the WDFC locus encode proteins that appear to have a role in immunity and/or fertility, two processes that are often associated with adaptive evolution. This study provides further evidence that the WFDC and SEMG loci have been under strong adaptive pressure within the short timescale of modern humans.

Loss and Gain of Function in SERPINB11: An Example of a Gene under Selection on Standing Variation, with Implications for Host-Pathogen Interactions

Serine protease inhibitors (SERPINs) are crucial in the regulation of diverse biological processes including inflammation and immune response. SERPINB11, located in the 18q21 gene cluster, is a polymorphic gene/pseudogene coding for a non-inhibitory SERPIN. In a genome-wide scan for recent selection, SERPINB11 was identified as a potential candidate gene for adaptive evolution in Yoruba. The present study sought a better understanding of the evolutionary history of SERPINB11, with special focus on evaluating its selective signature. Through the resequencing of coding and noncoding regions of SERPINB11 in 20 Yorubans and analyzing primate orthologous sequences, we identified a full-length SERPINB11 variant encoding a non-inhibitory SERPIN as the putative candidate of selection – probably driven to higher frequencies by an adaptive response using preexisting variation. In addition, we detected contrasting evolutionary features of SERPINB11 in primates: While primate phylogeny as a whole is under purifying selection, the human lineage shows evidence of positive selection in a few codons, all associated with the active SERPINB11. Comparative modeling studies suggest that positively selected codons reduce SERPINB11s ability to undergo the conformational changes typical of inhibitory SERPINs – suggesting that it is evolving towards a new non-inhibitory function in humans. Significant correlations between SERPINB11 variants and the environmental variables, pastoralism and pathogen richness, have led us to propose a selective advantage through host-pathogen interactions, possibly linked to an adaptive response combating the emergence of infectious diseases in recent human evolution. This work represents the first description of a resurrected gene in humans, and may well exemplify selection on standing variation triggered by drastic ecological shifts.

Birth-and-Death of KLK3 and KLK2 in Primates: Evolution Driven by Reproductive Biology

The kallikrein (KLK) gene family comprises the largest uninterrupted locus of serine proteases in the human genome and represents a notable case for studying the evolutionary fate of duplicated genes. In primates, a recent duplication event gave rise to KLK2 and KLK3, both encoding essential proteins for the cascade of seminal plasma liquefaction. We reconstructed the evolutionary history of KLK2 and KLK3 by comparative analysis of the orthologous sequences from 22 primate species, calculated dN/dS ratios, and addressed the hypothesis of coevolution with their substrates, the semenogelins (SEMG1 and SEMG2). Our findings support the placement of the KLK2–KLK3 duplication in the Catarrhini ancestor and unveil the frequent loss of KLK2 throughout primate evolution by different genomic mechanisms, including unequal crossing-over, deletions, and pseudogenization. We provide evidences for an adaptive evolution of KLK3 toward an expanded enzymatic spectrum, with an effect on the hydrolysis of semen coagulum. Furthermore, we found associations between mating system, the number of SEMG repeat units, and the number of functional KLK2 and KLK3, suggesting complex evolutionary dynamics shaped by reproductive biology.

Evolutionary Constraints in the β-Globin Cluster: The Signature of Purifying Selection at the δ-Globin (HBD) Locus and Its Role in Developmental Gene Regulation

Human hemoglobins, the oxygen carriers in the blood, are composed by two α-like and two β-like globin monomers. The β-globin gene cluster located at 11p15.5 comprises one pseudogene and five genes whose expression undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition. HBD encodes the δ-globin chain of the minor adult hemoglobin (HbA2), which is assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. In this study, we sought a detailed portrait of the genetic variation within the β-globin cluster in a large human population panel from different geographic backgrounds. We resequenced the coding and noncoding regions of the two adult β-globin genes (HBD and HBB) in European and African populations, and analyzed the data from the β-globin cluster (HBE, HBG2, HBG1, HBBP1, HBD, and HBB) in 1,092 individuals representing 14 populations sequenced as part of the 1000 Genomes Project. Additionally, we assessed the diversity levels in nonhuman primates using chimpanzee sequence data provided by the PanMap Project. Comprehensive analyses, based on classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and nonredundant. Moreover, in the light of recent studies on the chromatin conformation of the β-globin cluster, we present evidence sustaining that the strong functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but instead are likely due to a regulatory role in ontogenic switches of gene expression.