Reflexiones sobre la ingeniería genética: a propósito del nacimiento de gemelas sometidas a edición génica / Considerations on genetic engineering: regarding the birth of twins subjected to gene edition

Resumen En este ensayo se analizan las implicaciones bioéticas de la reciente manipulación genética en embriones humanos con CRISPR-Cas9 para eliminar el gen CCR5 y el nacimiento de dos gemelas discordantes. El experimento se divulgó en medios sociales. Los principales problemas bioéticos identificados son la justificación del modelo, el proceso de consentimiento informado y la falta de declaración de evidentes conflictos de interés. No se evaluaron apropiadamente las consecuencias del experimento sobre la vida de las gemelas nacidas como la afectación a su autonomía, los supuestos beneficios por recibir y los riesgos futuros de daño durante su vida. Habiendo manipulado la línea celular germinal, no se consideraron los efectos sobre su descendencia futura. Este tipo de acciones tiene un impacto negativo en la forma como la sociedad concibe la ciencia. La ingeniería genética debe reservarse al contexto experimental básico o bien como investigación cínica para la corrección de enfermedades conocidas graves de origen genético, bajo estricta supervisión regulatoria y bioética y de manera gradualista de acuerdo con el progreso de las técnicas de edición genética.

Abstract In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.

Haplotypes in CCR5-CCR2 , CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort / Los haplotipos en CCR5-CCR2 , CCL3 y CCL5 se asocian con resistencia natural a la infección por el HIV-1 en una cohorte colombiana

RESUMEN Introduction: Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. Objective: To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. Materials and methods: Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value <0.05 after Bonferroni correction was considered significant. Results: Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p'<0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). Conclusion: Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

ABSTRACT Introducción. Algunas variantes en genes que codifican los correceptores del HIV-1 y sus ligandos se han asociado individualmente a la resistencia natural frente a dicha infección. Sin embargo, su presencia simultánea ha sido poco estudiada. Objetivo. Evaluar la asociación de haplotipos individuales y multilocus en genes que codifican los correceptores virales CCR5 y CCR2 y sus ligandos CCL3 y CCL5 con la resistencia o la propensión a la infección por el HIV-1. Materiales y métodos. Nueve variantes en CCR5-CCR2, dos en CCL3 y dos en CCL5 fueron genotipificadas mediante reacción en cadena de la polimerasa de polimorfismos de longitud de fragmentos de restricción (Restriction Fragment Length Polymorphism-PCR-RFLP) en 35 individuos seropositivos (casos) y 49 seronegativos expuestos (controles) de Colombia. Los haplotipos se infirieron utilizando el programa Arlequín, y su frecuencia individual o combinada se comparó en los casos y los controles mediante la prueba de ji al cuadrado. Se consideró significativo un valor de p'<0,05 después de la corrección de Bonferroni. Resultados. La homocigosis del haplogrupo humano (HH) E estaba ausente en los controles y era frecuente en los casos, es decir, con tendencia hacia la propensión. Los haplotipos C-C y T-T en CCL3 se asociaron con la propensión (p'=0,016) y la resistencia (p'<0,0001), respectivamente. Por último, en el análisis multilocus, el haplotipo combinado formado por HHC en CCR5-CCR2, T-T en CCL3 y G-C en CCL5 se asoció con la resistencia (p'=0,006). Conclusión. Los resultados de este estudio sugieren que ciertas combinaciones específicas de variantes en los genes de una misma vía de señalización pueden definir un fenotipo resistente al HIV-1. Aunque el tamaño de la muestra era pequeño, las asociaciones estadísticamente significativas sugieren un efecto considerable; sin embargo, estos resultados deben validarse en cohortes de mayor tamaño.

[Evaluation of prevalence of delta 32 mutation in CCR5 gene in breast cancer patients in Rafsanjan city]

Chemokines and their receptors are expressed in different types of malignancies. CC chemokines MIP-1alpha [CCL3], MIP-1beta [CCL4] and RANTES [CCL5] is believed to be anti-tumor and also aid to the metastasis in tumor microenvironment. CCR2 and CCR5 are special G-protein receptors for these chemokines. Due to the important role of CCR5 chemokine receptor in tumor biology, this project is designed to examine delta 32 mutation in CCR5 gene regards breast cancer. This experimental study was performed during 2007-8 on delta healthy adults and 36 breast cancer patients by Gap-PCR. The demographic information also was collected by questionner and t-test Chi-square was used for statistical analysis of data. Our results showed that none of breast cancer patients had CCR5-delta 32 mutation while 3 [3%] cases of controls had heterozygotic form of this mutation. Our results showed that there is not any CCR5-delta 32 mutation in patients. Therefore, it appears that this mutation don't play any role in breast cancer

Study of CCR5 Gene polymorphism in rheumatoid arthritis

Chemokines are critical for the inflammatory process in autoimmune diseases such as rheumatoid arthritis [RA]. The chemokine receptor-5 [CCR5] mediates chemotaxis by CC chemokines and is expressed by lymphocytes with the phenotype and monocyte/macrophages. A 32bp deletion in the CCR5 [CCR5-delta 32 allele] abolishes receptor expression in homozygotes, while CCR5-delta 32 carriers express less receptor level than wild type homozygotes. This polymorphism is related to resistance to HIV-1 infection and progression to AIDS. It is hypothesized that CCR5-delta 32 allele may modulate the inflammatory response involved in rheumatoid arthritis and therefore may affect disease severity, susceptibility or both. In the present study 70 rheumatoid arthritis patients and 40 healthy individuals were genotyped. The frequency of CCR5-delta 32 allele was significantly higher in healthy individuals compared to rheumatoid arthritis patients [45% Vs 17%] respectively [p.value 0.033]. Homozygous delta 32 mutation was not detected in patients or controls No significant difference was found between CCR5-delta 32 carriers and wild type homozygotes regarding clinical or laboratory findings except for the tender joint count and rheumatoid factor positivity which was higher in wild type homozygotes [p.value 0.046 and 0.007 respectively]. Our data suggest that CCR5-dlta 32 carriers may partially protected against rheumatoid arthritis, and suggest CCR5 receptor as a candidate for targeted therapy in rheumatoid arthritis

Genetic landscape of the people of India: a canvas for disease gene exploration.

Analyses of frequency profiles of markers on disease or drug-response related genes in diverse populations are important for the dissection of common diseases. We report the results of analyses of data on 405 SNPs from 75 such genes and a 5.2 Mb chromosome, 22 genomic region in 1871 individuals from diverse 55 endogamous Indian populations. These include 32 large (>10 million individuals) and 23 isolated populations, representing a large fraction of the people of India. We observe high levels of genetic divergence between groups of populations that cluster largely on the basis of ethnicity and language. Indian populations not only overlap with the diversity of HapMap populations, but also contain population groups that are genetically distinct. These data and results are useful for addressing stratification and study design issues in complex traits especially for heterogeneous populations.

RANTES, MCP-1, CCR2, CCR5, CXCR1 and CXCR4 Gene Polymorphisms are not Associated with the Outcome of Hepatitis B Virus Infection: Results from a Large Scale Single Ethnic Population

Recovery from hepatitis B virus (HBV) infection depends on the cellular immune responses. Chemokines and their receptors play significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of genes for the chemokines and their receptors. Between March 2002 and February 2004, a total of 957 single ethnic Korean patients were enrolled into two different groups; "HBV clearance group" (n=350), who have recovered from HBV infection, and "HBV persistence group" (n=607), who were repeatedly HBsAg-positive. The HBV persistence group was subdivided into "inactive carrier" and "HBV progression group (chronic hepatitis and cirrhosis)". We assessed polymorphisms in regulated and normal T-cell expressed and secreted (RANTES) at position -403, monocyte chemoattractant protein-1 (MCP-1) at position -2518, CCR2 V64I, CCR5 -2459, CXCR1 S276T and CXCR4 I138I using single primer extension assay. Genotype distributions of the "HBV clearance versus persistence group" and "inactive carrier versus HBV progression group" were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with RANTES -403, MCP-1 -2518, CCR2 V64I, CCR5 -2459, CXCR1 S276T, and CXCR4 I138I polymorphisms. In addition, no association of analyzed SNPs with HBV disease progression was found.

Association between CCR5 Promoter Polymorphisms and Hepatitis B Virus Infection / 대한간학회지

BACKGROUND/AIMS: Immunogenetic factors may play a role in determining the susceptibility of an individual to viral infection. CCR5 promoter polymorphisms are known to be associated with HIV infection. However, there has been no report on the association between CCR5 promoter polymorphism and HBV infection. Therefore, we investigated the relationship between the CCR5 promoter polymorphism and HBV infection. METHODS: A total of 377 patients were classified into two groups according to their HBV infection status: (1)he spontaneous clearance group (SC); HBsAg (-), anti-HBc (+), anti-HBs (+) (2)he chronic HBsAg (+) carrier group (CC); HBsAg (+), anti-HBc (+), anti-HBs (-). CCR5 polymorphisms were detected by employing matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)- based SNP scoring assay, termed Restriction Fragment Mass Polymorphism (RFMP), which exploits the differences in molecular masses between the common allele and rare allele bases of interest. RESULTS: We found that the genotype frequencies of CCR5 A59029G significantly differed between the SC group (n=138) and CC group (n=239) (P<0.05). The CCR5 59029A allelic genotype was associated with an increased risks of chronic infection rather than spontaneous clearance (P=0.002), and the presence of the CCR5 59029G allele was significantly associated with the spontaneous clearance of HBV (P=0.001). Strong linkage disequilibrium between the CCR5-59029 and the CCR5-59353 polymorphic variants was identified. None of the 377 subjects had the CCR5-32 bp deletion mutation. CONCLUSIONS: The CCR5 promoter polymorphisms at position 59029 might play a role in the clearance of HBV infection. This primary experimental evidence needs further studies to clarify the clinical usefulness of CCR5 promoter polymorphisms as a target for the screening or treatment of HBV infection.

Prevalence of HIV-1 drug resistance in antiretroviral-naive pregnant Thai women.

HIV-1 drug resistance may limit the use of antiretrovirals when attempting to reduce the vertical transmission rate. Establishing the prevalence of the HIV-1 mutations associated with antiretroviral resistance in pregnant women will enable clinicians to maximize the chances of preventing vertical transmission. In order to determine the prevalence of HIV-1 resistant strains among antiretroviral-naive pregnant Thai women, the nucleotide sequences of the HIV-1 polymerase (pol) gene were evaluated. The plasma samples were collected from the women during the 34th week of pregnancy: numerous secondary mutations could be found in the reverse transcriptase (RT) and protease gene, while no primary mutations in the pol gene were found. The result also showed that by detecting the delta32bp deletion within the CCR 5 locus, it was evident that none of HIV-1 infected individuals had homozygous or heterozygous delta32bp deletions of the CCR5 gene; moreover, no CCR5 gene mutations were found in any individual.

Distribution of HIV-1 resistance-conferring polymorphic alleles SDF-1-3'A, CCR2-64I and CCR5-Delta32 in diverse populations of Andhra Pradesh, South India.

Polymorphic allelic variants of chemokine receptors CCR2 and CCR5, as well as of stromal-derived factor-1 SDF-1, the ligand for the chemokine receptor CXCR4, are known to have protective effects against HIV-1 infection and to be involved with delay in disease progression. We have studied the DNA polymorphisms at the loci that encode these proteins in 525 healthy individuals without any history of HIV-1 infection from 11 diverse populations of Andhra Pradesh, South India. The two protective alleles SDF-1-3'A and CCR2-64I at the SDF-1 and CCR2 loci, respectively, are present in all populations studied, although their frequencies differ considerably across populations (from 17% to 35% for the SDF-1-3'A allele, and from 3% to 17% for CCR2-64I). In contrast the CCR5-Delta32 allele is observed only in three populations (Yamani, Pathan and Kamma), all in low frequencies (i.e. 1% to 3%). The mean number of mutant alleles (for the three loci together) carried by each individual varies from 0.475 (in Vizag Brahmins) to 0.959 (in Bohra Muslims). The estimated relative hazard values for the populations, computed from the three-locus genotype data, are comparable to those from Africa and Southeast Asia, where AIDS is known to be widespread.

Frequency of CCR5 promotorpolymorphism affecting the rate of progression to AIDS [59029-G/A] in Kuwait

To estimate the frequencies of 59029-G and 59029-A alleles associated with slow and rapid progression to AIDS in Kuwaitis. The DNA was extracted from 109 blood samples of healthy unrelated Kuwaitis. The 59029-G/A polymorphism was detected by the polymerase chain reaction/restriction fragment length polymorphism [PCR-RFLP] test. The amplification of a 268-bp fragment of the CCR5 gene encompassing the site of the polymorphism was followed by digestion with restriction endonuclease Bsp 1286I and sizing the DNA fragments by agarose gel electrophoresis. Among 109 individuals genotyped, 10, 44 and 55 were 59029-A/A homozygous, 59029-G/G homozygous and 59029-G/A heterozygous, respectively. The frequency of the G allele was 0.66 [95% CI: 0.59-0.72] while the frequency of the A allele was 0.34 [95% CI: 0.28-0.41]. Conclusions: The frequency of the AIDS-'protective' 59029-G allele in Kuwaitis is significantly higher than the frequency of the 'rapid-progression' 59029-A allele. Moreover, the frequency of the 59029-G allele in Kuwaitis is among the highest reported. However, the difference from the 59029-G allele frequencies of other ethnic groups in some cases is not statistically significant

The first report of CCR5 delta 32 mutant in Thai injecting drug users.

CCR5, a chemokine receptor, is the principal coreceptor for macrophage-tropic HIV-1 which is the most important variant for viral transmission. It has been demonstrated that a homozygous genotype of a 32-bp deletion in CCR5 gene (delta32CCR5) shows a high degree of resistance to HIV-1 infection. To demonstrate that delta32CCR5 does exist in Thai natives, the CCR5 genotypes and allelic frequencies in 860 Thai injecting drug users (IDUs) were determined by PCR and DNA sequencing. Of these, six (0.7%) were CCR5/delta32CCR5 heterozygotes and no homozygote was found. The overall delta32CCR5 allelic frequency was 0.0035 and in HIV-1 seronegative (n = 490) and seropositive (n = 370) IDUs were 0.0051 and 0.0004, respectively, which were not significantly different (p = 0.3776). Here we report that the delta32CCR5 does exist in Thai IDUs as it is present in other human races. Such low allelic frequency may indicate that this mutation does not attribute a significant role in HIV-1 transmission in Thai IDUs.

Frecuencia del alelo mutado del receptor CCR-5 en individuos HIV-1 positivos y negativos en la Provincia del Chaco / Frequency of mutated allele of the CCR-5 receptor in HIV-1 positive and negative individuals in the Province of Chaco

Los individuos con alto riesgo a la infección por el virus HIV-1, portadores de una variante polimórfica del gen del receptor para b quimioquinas CCR-5 son resistentes a la infección por HIV-1. La mutación de CCR-5 es una deleción de 32 pares de bases dentro del gen resultando en una proteína truncada que no se expresa en la superficie. El genotipo homocigota está asociado a resistencia de la infección. Este alelo se encuentra más comúnmente en la población caucásica y no fue hallado en africanos o japoneses. El genotipo heterocigota disminuiría la entrada y replicación del HIV-1 a linfocitos T CD4 y macrófagos, asociándose de esta manera a una lenta progresión a SIDA. Con el objetivo de establecer la frecuencia del alelo mutado en individuos HIV-1(-) y HIV-1(+), en la población chaqueña, caucásica y con influencia hispana y guaraní, se estudiaron 118 individuos HIV-1(-) y 80 HIV-1(+). Una porción del gen CCR-5 fue amplificado por la técnica PCR, a partir de DNA genómico obtenido por el método de Salting out. Se observó en población HIV-1(-) un 2.5 por ciento del genotipo homocigota y 15.3 por ciento heterocigota, estos datos coinciden con los comunicados para otras poblaciones caucásicas. En la población HIV-1(+) no se halló el genotipo homocigota coincidiendo con otras publicaciones y la proporción de pacientes con la forma heterocigota fue de 2.5 por ciento, cifra menor a las reportadas por otros autores.

Epidemiology of HIV infection in the Republic of Korea

Since the first case of human immunodeficiency virus (HIV) infection in the Republic of Korea (ROK) was detected in 1985, 876 HIV-infected patients have been reported, as of December 1998. The male to female ratio was 6.8:1, and 87% of the patients were between 20 and 49 years of age. The major modes of transmission were sexual contacts, accounting for 86% of the cases (65% heterosexuals and 21% homosexuals). Transmission through blood and blood products accounted for 28 cases (3.2%), and vertical transmission for one case. No cases among intravenous drug abusers were reported. The seroprevalence among the blood donors was approximately one in 100,000. Subtypes A, B, C, D, E, and G of HIV-1 have been introduced into the ROK, and subtype B is the most predominant subtype. The frequency of the a deletion in the CCR5 gene, a coreceptor of HIV-1, was less than 1% among Koreans.