Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

Polymorphisms of apolipoprotein E in the Afro-descendant population of Buenaventura, Colombia / Polimorfismos del gen de la apopoliproteína E en la población afrodescendiente de Buenaventura, Colombia

ABSTRACT Objetives: To estimate the frequency distribution, both allelic and genotypic, of the APOE gene in the Afro-descendant population of Buenaventura, Colombia. Methods: Three hundred and forty-eight Afro-descendant individuals were analyzed and the APOE locus was genotyped by PCR-RFLP. The allelic and genotypic frequencies were established by direct counting and the Hardy-Weinberg equilibrium was evaluated through X2 test. The frequencies obtained in this study were compared with frequencies reported for other Colombian populations through the Fisher's exact test. Results: The following allelic frequencies were observed: E3, 70.8%; E4, 21.4%, and E2, 7.8%. The genotypic frequencies were: E3/E3, 51.1%; E3/E4, 27.3%; E2/E3, 12.1%; E4/E4, 6%; E2/E4, 3.5%, and E2/E2, 0%. The entire examined population was found in Hardy-Weinberg equilibrium (P = .074), and significant differences were found in the allele E4 when comparing this population with the Amerindian and mestizo populations of Bogotá, Quindío, Centro-Oriente, Valle del Cauca, Barranquilla and Medellín (P< 0.0345). Conclusions: The allelic frequencies observed in this study were significantly different from the frequencies reported in other Colombian populations. The high representativeness of the E4 and E2 alleles validates the hypothesis that there are micro-evolutionary processes that have been acting on their frequencies and could be associated with susceptibility to neuropsychiatric diseases such as Alzheimer's disease, metabolic alterations of fats and/or coronary artery disease.

RESUMEN Objetivos: Estimar la distribución de frecuencias tanto alélicas como genotípicas del gen APOE en la población afrodescendiente de Buenaventura, Colombia. Métodos: Mediante la técnica de PCR-RFLP's se analizaron 348 individuos no relacionados de esta ciudad. Se realizó el cálculo de frecuencias alélicas y genotípicas y se evaluó el equilibrio de Hardy-Weinberg mediante la prueba de la X2. Se compararon las frecuencias alélicas obtenidas en el presente estudio con otras poblaciones de Colombia mediante el test exacto de Fisher. Resultados: Se reportaron las siguientes frecuencias alélicas: E2, 7,8%; E3, 70,8%, y E4, 21,4%. Las frecuencias genotípicas fueron: E3/E3, 51,1%; E3/E4,27,3%; E4/E4,6%; E2/E3,12,1%; E2/E4, 3,5%, y E2/E2, 0%. La población total se encontró en equilibrio de Hardy-Weinberg (p = 0,074), y se hallaron diferencias significativas en el alelo E4 al comparar esta población con las amerindias y mestizas de Bogotá, Quindío, Centro-Oriente, Valle del Cauca, Barranquilla y Medellín (p < 0,0345). Conclusiones: Las frecuencias alélicas observadas fueron significativamente diferentes de las frecuencias reportadas en otras poblaciones de Colombia. La alta representatividad de los alelos E4 y E2 validan la hipótesis de que hay procesos microevolutivos que han venido actuando en sus frecuencias y pueden estar asociadas con susceptibilidad a enfermedades neuropsiquiátricas como la enfermedad de Alzheimer, alteraciones metabólicas de las grasas y/o enfermedad coronaria.

Apolipoprotein E4 influences growth and cognitive responses to micronutrient supplementation in shantytown children from northeast Brazil

OBJECTIVE: Apolipoprotein E4 may benefit children during early periods of life when the body is challenged by infection and nutritional decline. We examined whether apolipoprotein E4 affects intestinal barrier function, improving short-term growth and long-term cognitive outcomes in Brazilian shantytown children. METHODS: A total of 213 Brazilian shantytown children with below-median height-for-age z-scores (HAZ) received 200,000 IU of retinol (every four months), zinc (40 mg twice weekly), or both for one year, with half of each group receiving glutamine supplementation for 10 days. Height-for-age z-scores, weight-for-age z-scores, weight-forheight z-scores, and lactulose:mannitol ratios were assessed during the initial four months of treatment. An average of four years (range 1.4-6.6) later, the children underwent cognitive testing to evaluate non-verbal intelligence, coding, verbal fluency, verbal learning, and delayed verbal learning. Apolipoprotein E4 carriage was determined by PCR analysis for 144 children. RESULTS: Thirty-seven children were apolipoprotein E4(+), with an allele frequency of 13.9 percent. Significant associations were found for vitamin A and glutamine with intestinal barrier function. Apolipoprotein E4(+) children receiving glutamine presented significant positive Pearson correlations between the change in height-for-age z-scores over four months and delayed verbal learning, along with correlated changes over the same period in weight-for-age z-scores and weight-for-height z-scores associated with non-verbal intelligence quotients. There was a significant correlation between vitamin A supplementation of apolipoprotein E4(+) children and improved delta lactulose/mannitol. Apolipoprotein E4(-) children, regardless of intervention, exhibited negative Pearson correlations between the change in lactulose-to-mannitol ratio over four months and verbal learning and non-verbal intelligence. CONCLUSIONS: During development, apolipoprotein E4 may function concomitantly with gut-tropic nutrients to benefit immediate nutritional status, which can translate into better long-term cognitive outcomes.

Los factores de progresión del deterioro cognitivo leve a la enfermedad de Alzheimer / Roles in the progression from mild cognitive decline, to Alzheimer’s disease

El deterioro cognitivo y la demencia constituyen uno de los problemas de salud pública más importantes del siglo XXI. Dada su relación con la edad, constatamos en la última década un continuo incremento tanto en su incidencia como en su prevalencia, secundariamente al aumento progresivo de la longevidad en la población. Teniendo en cuenta los estudios de progresión realizados hasta la fecha y las cohortes de seguimiento del deterioro cognitivo podemos señalar algunos marcadores de evolución: edad avanzada, presencia de al menos un alelo de APOE 4 en el screenning genético, disminución del área hipocampal, puntuaciones bajas en test psicométricos, mayor compromiso o asociación a factores de riesgo vascular y antecedentes de familiares de primera línea con demencia, entre otros. Nos propusimos abordar el tema de los factores predictores.

Mild cognitive decline and dementia are both important health problems in the 21ist century . Given it’s connection with age, we have stated how, in the last decade a continuum increase in it’s incidence and prevalence is related with the progressive increase time of life. Having in consideration the studies of progression made up to date and the cohorts of following of mild cognitive decline, we can point out some markers of evolution, given: advanced age, the presence of at least one APOE 4 allele in genetic screening, diminished hippocampus area, low punctuations in psychometric tests, a major cardiovascular compromise and family background of first blood line relatives with dementia, among others. We had the purpose to approach the subject of the predictor factors with the main objective to offer updated information about the performed studies in connection with the topic.

Relationship of Nocturnal Sleep with Frontal Lobe Function in Mild Cognitive Impairment(MCI) Patients / 신경정신의학

OBJECTIVES: Nocturnal sleep disruption has been considered as a risk factor for cognitive impairment in the elderly. And the frontal lobe dysfunction was suggested to predict the progression to dementia. We aimed to illustrate the relationship of nocturnal sleep with frontal lobe function in mild cognitive impairment (MCI) patients. METHODS: Thirty MCI patients and 30 age- and sex-matched normal control (NC) subjects were selected. Frontal lobe function tests including Stroop Test, Similarity Test, Digit Span Test (DST), and Benton Visual Retention Test (BVRT) were administrated. Nocturnal polysomnography was done for each subject. RESULTS: There was no significant difference in the sleep parameters and diagnostic distributions of sleep disorders between the MCI and NC groups. In MCI patients, the mean hypopnea index (HI) of the ApoE4 positive group was higher than that of ApoE4 negative group. In the NC group, the wake time after sleep onset (WASO) was negatively correlated with the DST score (r=-0.545). In the MCI group, WASO tended to be negatively correlated with the Similarity Test score (r=-0.376, p=0.053), and slow wave sleep amount (SWS) was negatively correlated with the error score of BVRT (r=-0.489). CONCLUSION: Although there was no difference in the sleep quality and frequency of SAS between the MCI and NC groups, the severity of SDB was higher in MCI patients with ApoE4 compared to those without ApoE4. In the MCI group, the difficulty in maintaining sleep was associated with decreased executive function, and the decreased SWS was associated with impaired working memory. The relationship of nocturnal sleep with the frontal lobe function in MCI patients appears to be different from that of normal elderly subjects.

Effect of Truncated-ApoE4 Overexpression on tau Phosphorylation in Cultured N2a Cells / 华中科技大学学报（医学）（英德文版）

The carboxyl-terminal amino acids 272-299-truncated apoE4 (△272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (△272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (△272-299)cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-TapoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24-48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P＜0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (△272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (△272-299) might contribute the pathogenesis of Alzheimer disease.