Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor.

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Identification of c.1531C>T Pathogenic Variant in the CDH1 Gene as a Novel Germline Mutation of Hereditary Diffuse Gastric Cancer.

Germline pathogenic variants in the CDH1 gene are a well-established cause of hereditary diffuse gastric cancer (HDGC) syndrome. The aim of this study was to characterize CDH1 mutations associated with HDGC from Chile, a country with one of the highest incidence and mortality rates in the world for gastric cancer (GC). Here, we prospectively include probands with family history/early onset of diffuse-type of GC. The whole coding sequence of the CDH1 gene was sequenced from genomic DNA in all patients, and a multidisciplinary team managed each family member with a pathogenic sequence variant. Thirty-six cases were included (median age 44 years/male 50%). Twenty-seven (75%) patients had diffuse-type GC at ≤50 years of age and 19 (53%) had first or second-degree family members with a history of HDGC. Two cases (5.5%) carried a non-synonymous germline sequence variant in the CDH1 gene: (a) The c.88C>A missense variant was found in a family with three diffuse-type GC cases; and (b) c.1531C>T a nonsense pathogenic variant was identified in a 22-year-old proband with no previous family history of HDGC. Of note, six family members carry the same nonsense pathogenic variant. Prophylactic gastrectomy in the proband's sister revealed stage I signet-ring cell carcinoma. The finding of 1531C>T pathogenic variant in the CDH1 in proband with no previous family history of HDGC warrants further study to uncover familial clustering of disease in CDH1 negative patients. This finding may be particularly relevant in high incidence countries, such as the case in this report.

[Human papillomavirus testing in cervical cancer screening at a public health service of Santiago, Chile]. / Examen de detección de virus papiloma humano en el tamizaje de cáncer cervicouterino en un Servicio de Salud de Santiago, Chile.

BACKGROUND: Molecular techniques for human papillomavirus (HPV) detection have a good performance as screening tests and could be included in cervical cancer early detection programs. We conducted a population-based trial comparing HPV detection and Papanicolaou as primary screening tests, in a public health service in Santiago, Chile. AIM: To describe the experience of implementing this new molecular test and present the main results of the study. MATERIAL AND METHODS: Women aged 25 to 64 enrolled in three public health centers were invited to participate. In all women, samples were collected for Papanicolaou and HPV DNA testing, and naked-eye visual inspection of the cervix with acetic acid was performed. Women with any positive screening test were referred to the local area hospital for diagnostic confirmation with colposcopy and biopsy of suspicious lesions. RESULTS: Screening results were obtained for 8265 women, of whom 931 (11.3%) were positive to any test. The prevalence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was 1.1%; nine women had invasive cervical cancer. Sensitivities for the detection of CIN2+ were 22.1% (95% confidence interval (CI) 16.4-29.2) for Papanicolaou and 92.7% (95% CI 84.4-96.8) for HPV testing; specificities were 98.9% (95% CI 98.7-99.0) and 92.0% (95% CI 91.4-92.6) respectively. CONCLUSION: This experience showed that the implementation of a molecular test for cervical cancer screening is not a major challenge in Chile: it was well accepted by both the health team and the participants, and it may improve the effectiveness of the screening program.

[Effect of VKORC1 and CYP2C9 variants on dosage of oral anticoagulants in Chilean individuals]. / Efecto de las variantes de VKORC1 y CYP2C9 sobre la dosis de anticoagulantes orales en individuos chilenos.

BACKGROUND: The dose of oral anticoagulants (OAC) shows great variability among patients. Pharmacogenetic studies have shown that common variants in genes CYP2C9 (*2 and *3) and VKORC1 (-1639G>A) are associated with lower requirements of OAC. AIM: To study the association between average maintenance doses of oral anticoagulant therapy required to maintain a stable INR and CYP2C9 and VKORC1 gene variants in Chilean adults. MATERIAL AND METHODS: Prospective study of patients on anticoagulant treatment and with a stable international normalized ratio (INR) for prothrombin time for at least three months. Patients were classified as having high or low acenocoumarol or warfarin requirements. Peripheral blood DNA genotyping was performed by polymerase chain reaction and restriction fragment polymorphism or sequencing and electrophoresis. RESULTS: The study included 185 patients, 125 on acenocoumarol and 60 on warfarin. Patients with VKORC1-1639A allele were more likely to require lower doses of both drugs than patients with the G allele (Odds ratio [OR] for acenocoumarol 9.06, and OR for warfarin = 18.7). There was no association between CYP2C9*2 and*3 and acenocoumarol or warfarin requirements. CONCLUSIONS: There is an association between VKORC1-1639A variant and anticoagulant doses.

[Early detection of cervical cancer in Chile: time for change]. / Detección precoz del cáncer cervicouterino en Chile: tiempo para el cambio.

Mortality rates for cervical cancer (CC) in Chile are higher than those of developed countries and it has an unequal socioeconomic distribution. The recognition of human papilloma virus (HPV) as the causal agent of cervical cancer in the early 80's changed the prevention paradigms. Current goals are to prevent HPV infection by vaccination before the onset of sexual activity and to detect HPV infection in women older than 30 years. This article reviews CC prevention and early detection methods, discusses relevant evidence to support a change in Chile and presents an innovation proposal. A strategy of primary screening based on HPV detection followed by triage of HPV-positive women by colposcopy in primary care or by cytological or molecular reflex testing is proposed. Due to the existence in Chile of a well-organized nationwide CC prevention program, the replacement of a low-sensitivity screening test such as the Papanicolau test with a highly sensitive one such as HPV detection, could quickly improve the effectiveness of the program. The program also has a network of personnel qualified to conduct naked-eye inspections of the cervix, who could easily be trained to perform triage colposcopy. The incorporation of new prevention strategies could reduce the deaths of Chilean women and correct inequities.

[Pneumocystis jiroveci pneumonia: comparative study of cases in HIV-infected patients and immunocompromised non-HIV-infected patients]. / Características clínicas, diagnósticas y pronosticas de pacientes con neumonía por Pneumocystis jiroveci en individuos infectados por virus de inmunodeficiencia humana e individuos inmunocomprometidos por otra etiología: comparative study of cases in HIV-infected patients and immunocompromised non-HIV-infected patients.

BACKGROUND: Although P. jiroveci pneumonia affects immunocompromised (IC) patients of any etiology, clinical features and prognostic outcomes are different depending if they are patients with HIV infection or other causes of IC. OBJECTIVES: To compare clinical and laboratory features as well as outcomes of P. jiroveci pneumonia in HIV versus non-HIV patients. METHODS: Retrospective review of clinical records of HIV and non-HIV patients with P. jiroveci pneumonia managed at the Hospital Clínico Universidad Católica in Santiago, Chile, between 2005 and 2007. RESULTS: We included 28 HIV and 45 non-HIV patients with confirmed P. jiroveci pneumonia. The non-HIV population was older (65 vs 36,2 years, p < 0,01), had shorter duration of symptoms (7 [1-21] vs 14 [2-45] days, p < 0,01), required more invasive techniques (60 vs 21%, p < 0,01) and RT-PCR to confirm the diagnosis (93 vs 68%, p < 0,01), were more frequently treated at intensive care units (58 vs. 25%, p < 0,01) requiring artificial ventilation (56 vs 11%, p < 0,01), and had a higher attributable mortality (33% vs 0%, p < 0,01). CONCLUSIONS: Our study confirmed that P. jiroveci pneumonia in non-HIV IC patients is more severe, more difficult to diagnose and has higher mortality that in HIV patients. Therefore, it is mandatory to optimize diagnostic and therapeutic strategies for this patients group.

Screening trial of human papillomavirus for early detection of cervical cancer in Santiago, Chile.

Cervical cancer mortality in Chile is four times higher than in developed countries. We compared the accuracy of human papillomavirus (HPV) DNA testing and conventional Papanicolaou (Pap) testing to detect prevalent precancerous and cancerous lesions in the routine clinical practice of the public health system. Women aged 25 years and older residing in the area covered by three primary care centers of Santiago, Chile, were invited to participate. Eligible women received both HPV DNA (Hybrid Capture 2) and Pap testing. Women positive by either test (Pap: ASCUS+, HC2: RLU/CO ≥ 1.0) underwent colposcopy and biopsy, as did a sample of double-negative women with an abnormal cervix at visual inspection or with risk factors for cervical lesions. Crude and verification bias-corrected sensitivities and specificities were estimated. In total, 8,265 women (98.8% of eligible) had complete screening results. Of these, 10.7% were HPV positive, 1.7% were Pap positive and 1.1% were positive by both tests. In all, 931 (11.3%) women were screen-positive, of whom 94.3% attended colposcopy. Additionally, 295 control women were invited for colposcopy, of whom 78% attended. In all, 42 CIN2, 45 CIN3 and 9 cancers were identified. Verification bias-corrected sensitivity for CIN2+ (95% confidence interval) was 92.7% (84.4-96.8) for HPV and 22.1% (16.4-29.2) for Pap; corresponding specificities were 92.0% (91.4-92.6) and 98.9% (98.7-99.0). In conclusion, in routine clinical practice in a developing country, HPV testing was four times more sensitive for CIN2+ than Pap testing, identifying three times more CIN2+ lesions; HPV testing was easily implemented in our established cervical cancer prevention program.

HPV vaginal self-sampling among women non-adherent to Papanicolaou screening in Chile.

OBJECTIVE: To evaluate acceptance, preference and compliance with referral of vaginal self-sampling for the detection of Human papillomavirus (HPV) among women non-adherent to Papanicolaou (Pap) screening in Santiago, Chile. MATERIALS AND METHODS: Using multistage sampling we identified women aged 30-64 years who reported not receiving a Pap test in the previous three years and offered them Pap testing at the health center or vaginal self-sampling for HPV testing at home. Self-collected samples were analyzed with hybrid capture. All HPV+ women were referred for colposcopy, biopsy and treatment when needed. RESULTS: 1 254 eligible women were contacted; 86.5% performed self-sampling and 8.1% refused; 124 women were HPV+ (11.4%: 95%CI 9.6-13.5) of whom 85.5% attended colposcopy; 12 had CIN2+ (1.1%: 95 %CI 0.5-1.7). CONCLUSION: HPV vaginal self-sampling can be easily implemented in Chile and could improve coverage, successfully reaching women who drop out of the screening program.

[Basic concepts about paternity testing]. / Conceptos básicos sobre el estudio de paternidad.

Nowadays, the analysis of genetic markers is a very important and validated tool for the identification of individuals, and for paternity testing. To do so, highly variable regions of the human genome are analyzed, making it possible to obtain the genetic profile of an individual, and to distinguish between different individuals. The methodology used is basically the same all over the world, consisting in the analysis of 13 to 15 markers. To assign biological paternity the child must have inherited the characteristics from the alleged father in each of the genetic markers analyzed. This analysis achieves a certainty higher than with any other test, which is expressed as the probability of paternity. This probability has to be at least 99.9%, but greater probabilities are usually obtained, especially if the mother is included in the analysis. If the characteristics of two or more genetic markers from the alleged father are absent in the child, biological paternity is excluded.

[Introns 1 and 22 inversions and F8 gene sequencing for molecular diagnosis of hemophilia A in Chile]. / Análisis de las inversiones del intrón 1 y 22 y secuenciación del gen F8 para el diagnóstico genético-molecular de hemofilia A en Chile.

BACKGROUND: Hemophilia A is an inherited disorder caused by alterations in factor VIII gene (F8) located on the X-chromosome, the intron 22 inversion being the most common mutation. The rest are predominantly point mutations distributed along this large gene of 26 exons. AIM: To implement a molecular diagnostic test to detect mutations in the F8 gene in Chilean patients with Hemophilia A. MATERIAL AND METHODS: To validate the testing methods, we analyzed samples with intron 22 and intron 1 inversion, and with point mutations previously studied, as well as one subject without Hemophilia. We also studied unrelated Chilean patients with Hemophilia A and their female relatives for carrier testing. Intron 22 and intron 1 inversions were studied by long distance polymerase chain reaction (PCR) and point mutations by sequencing the coding and promoter regions of the F8 gene. RESULTS: The results obtained in all samples used for validation were concordant with those obtained previously. In the Chilean patients, the intron 22 inversion and point mutations previously described were observed. In 6 out of 9 patients with mild Hemophilia A we found the same mutation (Arg2159Cys) in exon 23, which has been described as prevalent in mild Hemophilia A. CONCLUSIONS: The analysis of intron 22 and intron 1 inversions, as well as of point mutations in the F8 gene will help us to confirm the diagnosis in patients with severe, moderate and mild Hemophilia A, and also it will allow us to perform carrier testing and to provide better genetic counseling.

[Laboratory diagnosis of Treponema pallidum infection in patients with early syphilis and neurosyphilis through a PCR-based test]. / Diagnóstico de la infección por Treponema pallidum en pacientes con sífilis temprana y neurosífilis mediante reacción de la polimerasa en cadena.

Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47 gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis.

Detection of a novel severe mutation affecting the CYP21A2 gene in a Chilean male with salt wasting congenital adrenal hyperplasia.

PURPOSE: 21-hydroxylase deficiency (21-OHD) is a congenital adrenal disease with more than 200 mutations published to date. The aim of this report is to describe a severe novel mutation of the CYP21A2 gene. METHOD: We describe a case of a 39-year-old male diagnosed with a salt wasting congenital adrenal hyperplasia (SWCAH) due to 21-OHD. The genetic testing was done using a combination of three methods (PCR XL, SALSA-MLPA, and bidirectional sequencing) and finally an in silico analysis. RESULTS: The genetic testing demonstrated three severe mutations of the CYP21A2 gene (p.Gln318*; c.290-13C>G; and p.Trp86*), being the last one a novel mutation not previously reported. The in silico modeling of the p.Trp86* (c.258G>A) showed a truncated CYP21A2 protein that loses all the main structural features required for activity, such as the HEM binding domain and the hormone binding site. CONCLUSION: We present an adult man with an SWCAH due to 21-OHD who carried three severe mutations of the CYP21A2 gene, one of them, p.Trp86* (c.258G>A) has not been previously described.

A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR.

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

A Thyroid Genetic Classifier Correctly Predicts Benign Nodules with Indeterminate Cytology: Two Independent, Multicenter, Prospective Validation Trials.

Background: Although most thyroid nodules with indeterminate cytology are benign, in most of the world, surgery remains as the most frequent diagnostic approach. We have previously reported a 10-gene thyroid genetic classifier, which accurately predicts benign thyroid nodules. The assay is a prototype diagnostic kit suitable for reference laboratory testing and could potentially avoid unnecessary diagnostic surgery in patients with indeterminate thyroid cytology. Methods: Classifier performance was tested in two independent, ethnically diverse, prospective multicenter trials (TGCT-1/Chile and TGCT-2/USA). A total of 4061 fine-needle aspirations were collected from 15 institutions, of which 897 (22%) were called indeterminate. The clinical site was blind to the classifier score and the clinical laboratory blind to the pathology report. A matched surgical pathology and valid classifier score was available for 270 samples. Results: Cohorts showed significant differences, including (i) clinical site patient source (academic, 43% and 97% for TGCT-1 and -2, respectively); (ii) ethnic diversity, with a greater proportion of the Hispanic population (40% vs. 3%) for TGCT-1 and a greater proportion of African American (11% vs. 0%) and Asian (10% vs. 1%) populations for TGCT-2; and (iii) tumor size (mean of 1.7 and 2.5 cm for TGCT-1 and -2, respectively). Overall, there were no differences in the histopathological profile between cohorts. Forty-one of 155 and 45 of 115 nodules were malignant (cancer prevalence of 26% and 39% for TGCT-1 and -2, respectively). The classifier predicted 37 of 41 and 41 of 45 malignant nodules, yielding a sensitivity of 90% [95% confidence interval; CI 77-97] and 91% [95% CI 79-98] for TGCT-1 and -2, respectively. One hundred one of 114 and 61 of 70 nodules were correctly predicted as benign, yielding a specificity of 89% [95% CI 82-94] and 87% [95% CI 77-94], respectively. The negative predictive values for TGCT-1 and TGCT-2 were 96% and 94%, respectively, whereas the positive predictive values were 74% and 82%, respectively. The overall accuracy for both cohorts was 89%. Conclusions: Clinical validation of the classifier demonstrates equivalent performance in two independent and ethnically diverse cohorts, accurately predicting benign thyroid nodules that can undergo surveillance as an alternative to diagnostic surgery.

[Assessment of the Amplicor PCR for the detection of Mycobacterium tuberculosis in smear negative respiratory and non respiratory specimens: a retrospective analysis]. / Análisis retrospectivo del rendimiento de Amplicor-PCR para la detección de Mycobacterium tuberculosis en muestras respiratorias y no respiratorias con baciloscopia negativa.

BACKGROUND: Commercial polymerase chain reaction (PCR) kits are widely accepted for analysis of smear positive respiratory specimens, but the sensitivity is variable for smear negative ones. OBJECTIVE: To assess the PCR method usefulness in smear negative respiratory and non respiratory specimens. METHODS: We compared the PCR results (AMPLICOR MTB test, Roche) of 235 specimens subjected to culture in Loewenstein-Jensen agar (as the gold standard). RESULTS: 181 (76%) were respiratory and 54 (24%) extra-respiratory specimens. The sensitivity was 88%) and 50%>, respectively, specificity and PPV was 100%> in both cases. NPV was 99.4%> in respiratory specimens and 96.1% in non-respiratory specimens. CONCLUSIONS: The good performance of this PCR in smear negative respiratory specimens allows the clinician to take decisions based on the result of this exam. In extra-respiratory specimens the contribution is important only when the PCR result is positive.

Genetic testing for indeterminate thyroid cytology: review and meta-analysis.

Thyroid cancer is the most frequent endocrine malignancy, and its incidence is increasing. A current limitation of cytological evaluation of thyroid nodules is that 20-25% are reported as indeterminate. Therefore, an important challenge for clinicians is to determine whether an indeterminate nodule is malignant, and should undergo surgery, or benign, and should be recommended to follow-up. The emergence of precision medicine has offered a valuable solution for this problem, with four tests currently available for the molecular diagnosis of indeterminate cytologies. However, efforts to critically analyze the quality of the accumulated evidence are scarce. This systematic review and meta-analysis is aimed to contribute to a better knowledge about the four available molecular tests, their technical characteristics, clinical performance, and ultimately to help clinicians to make better decisions to provide the best care options possible. For this purpose, we address three critical topics: (i) the proper theoretical accuracy, considering the intended clinical use of the test (rule-in vs rule-out) and the impact on clinical decisions; (ii) the quality of the evidence reported for each test (iii) and how accurate and effective have the tests proved to be after their clinical use. Together with the upcoming evidence, this work provides significant and useful information for healthcare system decision-makers to consider the use of molecular testing as a public health need, avoiding unnecessary surgical risks and costs.

HPV16/18 genotyping for the triage of HPV positive women in primary cervical cancer screening in Chile.

BACKGROUND: We previously conducted a population-based screening trial of high-risk human papillomavirus (hrHPV) testing and conventional cytology, demonstrating higher sensitivity (92.7 % vs 22.1 % for CIN2+) but lower positive predictive value (10.5 % vs 23.9 %) of hrHPV testing. Here we report the performance of HPV16/18 genotyping to triage the hrHPV positive participants. METHODS: Women aged 25 years and older received hrHPV (Hybrid Capture 2) and Papanicolaou testing; positives by either test underwent colposcopy and directed biopsy, as did a sample of double-negatives. hrHPV positive women were reflex-tested with HPV16/18 genotyping (Digene HPV Genotyping PS Test). RESULTS: Among the 8,265 participants, 10.7 % were hrHPV positive, 1.7 % had ASCUS+ cytology, 1.2 % had CIN2+; 776 (88 %) hrHPV positive women had complete results, of whom 38.8 % were positive for HPV16 (24.0 %), HPV18 (9.7 %) or both (5.1 %). CIN2+ prevalence in HPV16/18 positive women (16.3 %, 95 % CI 12.3-20.9) was twice that of HPV16/18 negative women (8.0 %, 95 % CI 5.7-10.8). HPV16/18 genotyping identified 40.5 % of CIN2, 66.7 % of CIN3 and 75.0 % of cancers. Compared to hrHPV screening alone, HPV16/18 triage significantly reduced the referral rate (10.7 % vs 3.7 %) and the number of colposcopies required to detect one CIN2+ (9 vs 6). When HPV16/18 negative women with baseline ASCUS+ cytology were also colposcopied, an additional 14 % of CIN2+ was identified; referral increased slightly to 4.2 %. CONCLUSIONS: HPV16/18 triage effectively stratified hrHPV positive women by their risk of high-grade lesions. HPV16/18 positive women must be referred immediately; referral could be deferred in HPV16/18 negative women given the slower progression of non-HPV16/18 lesions, however, they will require active follow-up.

A Deletion of More than 800 kb Is the Most Recurrent Mutation in Chilean Patients with SHOX Gene Defects.

BACKGROUND: Deletions in the SHOX gene are the most frequent genetic cause of Leri-Weill syndrome and Langer mesomelic dysplasia, which are also present in idiopathic short stature. AIM: To describe the molecular and clinical findings observed in 23 of 45 non-consanguineous Chilean patients with different phenotypes related to SHOX deficiency. METHODS: Multiplex ligation-dependent probe amplification was used to detect the deletions; the SHOX coding region and deletion-flanking areas were sequenced to identify point mutations and single-nucleotide polymorphisms (SNPs). RESULTS: The main genetic defects identified in 21 patients consisted of deletions; one of them, a large deletion of >800 kb, was found in 8 patients. Also, a smaller deletion of >350 kb was observed in 4 patients. Although we could not precisely determine the deletion breakpoint, we were able to identify a common haplotype in 7 of the 8 patients with the larger deletion based on 22 informative SNPs. CONCLUSION: These results suggest that the large deletion-bearing allele has a common ancestor and was either introduced by European immigrants or had originated in our Amerindian population. This study allowed us to identify one recurrent deletion in Chilean patients; also, it contributed to expanding our knowledge about the genetic background of our population.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

A novel insertion in the FOXL2 gene in a Chilean patient with blepharophimosis ptosis epicanthus inversus syndrome type I.

OBJECTIVE: A novel insertion in the forkhead transcription factor 2 (FOXL2) was identified in a Chilean patient with blepharophimosis, ptosis, and epicanthus inversus syndrome associated with premature ovarian failure (BPES type I). A clinical and molecular characterization of a patient with BPES type I was performed. METHOD: We present a 16-year-old adolescent girl with surgically treated blepharophimosis, ptosis, and epicanthus inversus that was associated with delayed puberty and secondary amenorrhea at the age of 15, indicators that suggested that the patient had BPES type I. The FOXL2 gene was analyzed by sequencing its coding region. RESULTS: The sequence analysis of the FOXL2 gene revealed a novel heterozygous mutation: an 11 bp duplication (c.901_911dup11) that was predicted to encode a truncated protein (p.Pro305Argfs*54). CONCLUSIONS: A novel out-of-frame duplication following the polyalanine domain in the FOXL2 gene was identified in a Chilean patient with BPES type I. This study characterized the molecular alterations in FOXL2 and confirmed the diagnosis, thereby providing information to allow for improved genetic counseling for the patient and her family.