Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be disease causing, with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser causador de doenças, com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing", with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Investigation on the elemental profiles of lip cosmetic products: Concentrations, distribution and assessment of potential carcinogenic and non-carcinogenic human health risk for consumer safety.

Metal contamination of lip care products can cause potential adverse effects for consumers, hence assessment of human health risks associated with the consumption of these products is inevitable to ensure the consumers' safety. In the presented study, the profiles of 18 elements in 37 of the most popular lip cosmetic products, of various types and brands, sold in the Saudi Arabian markets, were investigated and their associated potential carcinogenic and non-carcinogenic human health risks were assessed. The metal concentrations were determined using inductively coupled plasma mass spectrometry preceded by microwave digestion for sample preparation. In general, the concentrations of the investigated metals were lower than the safe permissible limits with the exception of Cd, Pb and Hg. The results found that Cd was regarded as the primary metal contaminant present in the analyzed lip products contributing to 66.3% of the total determined carcinogenic health risk. Overall, however it was observed that there was no significant non-carcinogenic (hazard index < 1) or carcinogenic (RiskT < 10-4) health risks associated with the use of the investigated lip products. Although all the calculated values in this study were within the acceptable limits, special attention should be taken in order to prioritize minimizing the trace metals in lip products, especially for Cd, Pb, Ti and Hg. This study could provide vital data needed to ascertain the degree to which heavy metal exposure through cosmetics is prevalent.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Cohort retrospective study: the neutrophil to lymphocyte ratio as an independent predictor of outcomes at the presentation of the multi-trauma patient.

BACKGROUND: Although the association of neutrophil to lymphocyte ratio (NLR) with mortality in trauma patients has recently been shown, there is a paucity of research on the association with other outcomes. Recent studies suggest that the NLR has a predictive value of mortality in trauma patients during various times of admission. This study aimed to determine the prognostic impact of NLR at the presentation in critically ill trauma patients. METHODS: A retrospective cohort study of adult trauma patients between July 2017 and November 2017 in Tishreen Hospital. All patients who had arrived at the emergency department with multi-trauma injury within the age category (14-80 years) were included in this analysis. The prophetical capability of NLR on mortality was assessed by the receiver operative characteristics (ROC) curve. To identify the impact of the NLR on survival, a separate log-rank test was used. Multivariable Cox proportional hazard modeling was used to identify independent predictors of mortality. RESULTS: Throughout the time of the study, 566 patients met the inclusion criteria. Of these, 98.8% were male, 75.8% sustained penetrating trauma, and median age [IQR25-IQR75] was 26 [23-32]. Ninety-seven patients (17.1%) had major trauma, with an Injury Severity Score (ISS) ≥ 15. Using the ROC curve analyses hospitalization day 1, optimal NLR cutoff values of 4.00 were calculated by maximizing the Youden index. Kaplan-Meier curves revealed an NLR greater than or equal to these cutoff values as a marker for increased in-hospital mortality (p = 0.020, log-rank test). The Cox regression model demonstrated significant collinearity among the predictive variables (all VIF results < 2). Only ISS > 15 has a significant statistical relation with elevated NLR on day 1 (p = 0.010). CONCLUSIONS: Elevated NLR on day 1 has high predictive power for overall survival during the first 30 days after trauma, but it was not independent of other factors.

Prenatal and early-life predictors of atopy and allergic disease in Canadian children: results of the Family Atherosclerosis Monitoring In earLY life (FAMILY) Study.

Prenatal and early-life environmental exposures play a key role in the development of atopy and allergic disease. The Family Atherosclerosis Monitoring In earLY life Study is a general, population-based Canadian birth cohort that prospectively evaluated prenatal and early-life traits and their association with atopy and/or allergic disease. The study population included 901 babies, 857 mothers and 530 fathers. Prenatal and postnatal risk factors were evaluated through questionnaires collected during the antenatal period and at 1 year. The end points of atopy and allergic diseases in infants were evaluated through questionnaires and skin prick testing. Key outcomes included atopy (24.5%), food allergy (17.5%), cow's milk allergy (4.8%), wheezing (18.6%) and eczema (16%). The association between infant antibiotic exposure [odds ratio (OR): 2.04, 95% confidence interval (CI): 1.45-2.88] and increased atopy was noted in the multivariate analysis, whereas prenatal maternal exposure to dogs (OR: 0.60, 95% CI: 0.42-0.84) and acetaminophen (OR: 0.68, 95% CI: 0.51-0.92) was associated with decreased atopy. This population-based birth cohort in Canada demonstrated high rates of atopy, food allergy, wheezing and eczema. Several previously reported and some novel prenatal and postnatal exposures were associated with atopy and allergic diseases at 1 year of age.