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Background The outbreak of coronavirus disease 2019 (COVID-19) has emerged as a serious public health emergency of global concern. Angiotensin converting enzyme 2 (ACE2) peptidase domain is important for the cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Germline variants in ACE2 peptidase domain may influence the susceptibility for SARS-CoV-2 infection and disease severity in the host population. ACE2 genetic analysis among Caucasians showed inconclusive results. This is the first Asian study investigating the contribution of ACE2 germline variants to SARS-CoV-2 infection in Pakistani population. Methods In total, 442 individuals, including SARS-CoV-2-positive (n=225) and SARS-CoV-2-negative (n=217) were screened for germline variants in ACE2peptidase domain (exons 2, 3, 9, and 10) using high resolution melting and denaturing high-performance liquid chromatography analyses followed by DNA sequencing of variant fragments. The identified variant was analyzed by in silico tools for potential effect on ACE2 protein. Results A missense variant, p.Lys26Arg, was identified in one SARS-CoV-2-positive (1/225; 0.4%) and three SARS-CoV-2-negative (3/217; 1.4%) individuals. No significant difference in the minor allele frequency of this variant was found among SARS-CoV-2-positive and SARS-CoV-2-negative individuals (1/313; 0.3% versus 3/328; 0.9%; P=0.624), respectively. The SARS-CoV-2-positive patient carrying p.Lys26Arg showed mild COVID-19 disease symptoms. It was predicted as benign variant by in silico tool. No variant was detected in ACE2 residues important for binding of SARS-CoV-2 spike protein. Conclusion The p.Lys26Arg variant may have no association with SARS-CoV-2 susceptibility in Pakistani population. Whole ACE2 gene screening is warranted to clarify its role in SARS-CoV-2 infection.

The Fanconi anemia complementation group M (FANCM) gene is a potential candidate for breast/ovarian cancer susceptibility in European populations. Here, we examined the contribution of FANCM germline variants to hereditary breast and/or ovarian cancer in Pakistan. Comprehensive FANCM variant screening was performed in 201 BRCA1 and BRCA2 (BRCA1/2) negative Pakistani patients with and without triple-negative breast cancer (TNBC) and/or ovarian cancer, using denaturing high-performance liquid chromatography analysis (DHPLC) followed by DNA sequencing. Novel variants were tested for their potential effect on protein function using in silico tools. Reverse transcription (RT)-PCR analysis of RNA extracted from one deletion/insertion (delins) variant (p.K1780delinsNGIT) carrier and three non-carriers was performed to evaluate the impact of this variant on splicing. Furthermore, potentially functional variants were evaluated in 200 healthy female controls. A missense variant (p.V1857M) was identified in a 50-year-old TNBC patient with a family history of breast cancer. It was also identified in the index patient´s daughter, who was diagnosed with osteosarcoma at 15 years of age. Further, one delins variant (p.K1780delinsNGIT) was identified in a 45-year-old non-TNBC patient, but not detected in her brother, who was diagnosed with Hodgkin's lymphoma at 38 years of age. Based on in silico and RNA analyses, p.V1857M and p.K1780delinsNGIT were predicted as variants of uncertain significance (VUS), respectively. Both variants were absent in 200 healthy controls. Our findings suggest a marginal contribution of FANCM variants to hereditary breast/ovarian cancer in Pakistan, which need to be confirmed in larger studies.

Oxidoreductases are widely recognized for their capability to degrade phenolic pollutants and versatile. However, the lack of enzyme stability makes this technique unrealistic for industrial applications. In order to enhance their catalytic activity, stability and reusability, oxidoreductases namely laccases and peroxidases were entrapped in sol–gel silica and their catalytic activities were measured by an enzymatic assay using 2,6-dimethoxyphenol and guaiacol as substrates, respectively. The sol–gel silica matrices acted as a polymeric framework around the enzyme is a promising tool for improving enzyme stability. After entrapment, the catalytic activity and stability of sol–gel laccase and peroxidase toward pH, temperature and storage duration remarkably enhanced.

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly hearing loss in mice and humans. Two forms of hearing loss are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing, and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense, and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used minigene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA splicing, and highlights the need to investigate the effect of coding variants on RNA splicing.

A computational technique to simulate turbulent and vegetated flow in a rectangular open channel was investigated. Reynolds stress model was implemented to the circular vegetation patch flow configuration for the investigation of flow properties and turbulence characteristics. A finite volume-based model was developed using a three-dimensional (3-D) numerical code FLUENT and the pre-processor Geometry and Mesh Building Intelligent Toolkit. The model was first validated and then used for simulation purpose. Vertical distribution of mean stream-wise velocities was computed at typical locations. A deceleration in the magnitude of stream-wise velocities was observed within the vegetation patch zone. Minimum values of velocity magnitude were observed directly behind the vegetation structures. Turbulence characteristics in the form of Reynolds stresses and turbulent kinetic energies investigated by this model showed that turbulence was greater in the regions of vegetation patch. This Reynolds stress modeling formulation has shown to be capable of capturing important mean flow and turbulence characteristics in the configuration considered.

Reservoirs play a strategic role in the rapid monetary growth of the world by providing numerous benefits. However, the reduction in appropriate sites along with environmental and social apprehensions has resulted in curtailment of new reservoirs around the world in twenty-first century. There is a potential of benefits available from existing reservoirs which can be best capitalized through their optimized operation. Reservoirs Operation Optimization considering Sediment Evacuation (RESOOSE), recently developed model which combines multiple reservoirs operation and sediment evacuation with Genetic Algorithm based optimization module, has been used in the study. The objective of the study was to optimize the irrigation deficit through cascade reservoirs with consideration to hydropower, sediment evacuation and flood damages reduction benefits. The RESOOSE model was applied to optimize the irrigation deficits of Tarbela and Diamer Basha Reservoirs in Pakistan using developed objective function. The article computed and compared the benefits of optimized and existing rule curves. The hydropower benefits of 36.92 Billion Kw, sediment evacuation benefits of 21.534 Million m3 and flood damages of 616.19 Million US$ due to existing rule curves were considered as minimum benefits for achieving the optimized rule curves to minimize irrigation deficits. The developed optimized rule curves reduced the irrigation shortages of case study reservoirs from 6.9 to 5.8 Billion m3 (16% enhancement) annually as compared to existing rule curves. The optimized rule curves minimized the irrigation deficits by maintaining the existing benefits and without lowering the minimum operating levels of case study reservoirs. The study suggests change in existing rule curves of Tarbela and Diamer Basha Reservoirs due to less irrigation shortages. The RESOOSE model can be applied to other cascade reservoirs for optimizing the rule curves.

Background Pathogenic germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers worldwide. To refine the spectrum of BRCA1/2 mutations and to accurately estimate the prevalence of mutation in the Pakistani population, we studied 539 breast cancer patients selected for family history and age of diagnosis. Methods Comprehensive screening for BRCA1/2 germline mutations was performed using state-of-the-art technologies. Results A total of 133 deleterious mutations were identified in 539 families (24.7%), comprising 110 in BRCA1 and 23 in BRCA2. The prevalence of BRCA1/2 small-range mutations and large genomic rearrangements was 55.4% (36/65) for families with breast and ovarian cancer, 27.4% (67/244) for families with two or more cases of breast cancer, 18.5% (5/27) for families with male breast cancer, and 12.3% (25/203) for families with a single case of early-onset breast cancer. Nine mutations were specific to the Pakistani population. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% (75/110) and 34.8% (8/23) of all identified mutations in BRCA1 and BRCA2, respectively. Most of these mutations were exclusive to a specific ethnic group and may result from founder effects. Conclusions Our findings show that BRCA1/2 mutations account for one in four cases of hereditary breast/ovarian cancer, one in five cases of male breast cancer, and one in eight cases of early-onset breast cancer in Pakistan. Our study suggests genetic testing of an extended panel of 21 recurrent BRCA1/2 mutations for appropriately selected patients and their families in Pakistan.

The paper develops a new methodology and RESOOSE model for optimizing operation of multiple reservoirs considering sediment evacuation for multiple objectives consisting of maximization of irrigation, hydropower, storage conservation and minimization of flood damages. The optimization and sediment evacuation modules of the model are based on Genetic Algorithm and Tsinghua equation respectively. According to the criteria developed for selection of reservoirs, three reservoirs on Indus River of Pakistan have been selected. The model has been calibrated and validated for Tarbela and Chashma Reservoirs. The calibrated model has been applied to Diamer Basha (proposed) and Tarbela Reservoirs. Eight scenarios comprising of combined and individual objectives were developed. The optimized results of different scenarios have been compared with existing rule curves. The optimization results reveal that objective function for hydropower, irrigation, and storage conservation benefits would be enhanced by 11, 13 and 84 % respectively while flood damages would reduce by 80 %. Similarly the optimized rule curves will increase the benefits up to 579, 960, 533 and 648 million US$ per year (on 2012 prices) for all the four components respectively. The maximum value of US$ 977 million per year has been found for the scenario 5 which gives equal priority weight to all the four sectors. The developed model can be applied to other two reservoirs (in series) for optimization of rule curves.

Pathogenic BRCA1/2 germline mutations confer high risks of breast and ovarian cancer to women of European ancestry. Characterization of BRCA1/2 mutations in other ethnic groups is also medically important. We comprehensively screened 68 Colombian breast/ovarian cancer families for small-range mutations, 221 families for large-genomic rearrangements, and 1,022 unselected breast cancer cases for Colombian founder mutations in BRCA1/2. The risk of cancer among relatives of mutation carriers and the mutation penetrance were estimated by survival analysis. Identified BRCA2 mutations included 6310delGA and the recurrent 1991del4 mutations. A novel large BRCA2 deletion was found in 0.9% of the screened families. Among unselected breast cancer cases, 3.3% tested positive for BRCA1/3450del4, 2.2% for BRCA1/A1708E, 1.1% for BRCA2/3034del4, and 0.4% for BRCA2/1991del4. Female relatives of carriers of BRCA1/2 founder mutations showed a 5.90 times higher risk of breast cancer, when the woman herself carried a BRCA1 mutation compared to a non-carrier (95% CI 2.01–17.3). The estimated cumulative risk of breast cancer by age 70 years for BRCA1 mutations carriers was 14% (95% CI 5–38) compared to 3% for the general Colombian population (relative risk of breast cancer 4.05). Together with known founder mutations, reported novel variants may ease a cost-effective BRCA1/2 screening in women with Colombian ancestry.

RAD51C plays a key role in homologous recombination-mediated DNA repair and maintenance of genomic stability. Biallelic RAD51C mutations cause Fanconi anemia, and monoallelic mutations predispose women to breast and ovarian cancer. Genetic variability of RAD51C and its impact in Asian populations have been poorly studied. Here, we report the results of comprehensive mutational screening of the RAD51C gene in 348 BRCA1/2-negative breast and/or ovarian cancer patients from Pakistan. Mutation analysis of the complete RAD51C-coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments. Three novel protein-truncating mutations, c.204T>A, c.225T>G, and c.701C>G, were identified. c.204T>A was found in one out of 22 (4.5 %) early-onset (≤45 years of age) ovarian cancer patients and c.225T>G in one out of 119 (0.8 %) patients from breast cancer only families. c.701C>G was found in a 60-year-old control with no family history of breast/ovarian cancer. Furthermore, three novel in silico-predicted potentially functional mutations, a missense mutation, c.873T>G, a variant in 5′UTR, c.1-34T>G, and a recurrent intronic variant, c.965+21A>G, were identified. The missense mutation was observed in a patient with bilateral breast cancer from a breast and ovarian cancer family (HBOC), the 5′UTR variant was noted in an early-onset breast cancer patient, and the intronic variant in one early-onset breast cancer patient and one ovarian cancer patient from a HBOC family. Five of the six mutations described were not detected in 400 healthy controls. These findings suggest that RAD51C plays a marginal role in breast and ovarian cancer predisposition in Pakistan. Reliable estimation of the clinical implications of carrying a deleterious RAD51C mutation will require identification of additional mutation-positive patients/families.

Dams are strategic assets as they provide water for irrigation, hydropower generation and control floods for rapid economic growth. However, due to lack of suitable sites as well as socio-environmental concerns, the development of new reservoirs has reduced, which necessitates better management of existing ones with consideration to sediment removal for storage conservation. This study presents an analysis of sediment management options available in the literature for Tarbela Reservoir. RESCON model has been used for technical and economical viability of flushing, dredging, trucking and hydrosuction processes. It is found that dredging and trucking are technically feasible but economically non-feasible, whereas, hydrosuction is technically non-feasible for Tarbela Reservoir. Reservoir flushing has been found technically and economically viable for this dam. HEC-RAS model has been used in the present study for carrying out Tarbela Reservoir sedimentation analysis to evaluate the options of: (i) sediment management through appropriate reservoir operation and (ii) constructing the proposed multipurpose Diamer Basha Dam, located at the upstream. The simulations lead to the conclusion that both the options are feasible. The reservoir storage depletion can be reduced and its life can be extended up to 27 years by lowering the minimum operation level from 457 to 401 m. The storage conservation may enhance the irrigation benefits and will reduce the devastations due to floods. Further studies are recommended to envisage the impact of high sediment outflows on turbines, tunnels and downstream reaches for Tarbela Reservoir operation at lower minimum operating levels and flushing.

Background Women harboring BRCA1/2 germline mutations have high lifetime risk of developing breast/ovarian cancer. The recommendation to pursue BRCA1/2 testing is based on patient’s family history of breast/ovarian cancer, age of disease-onset and/or pathologic parameters of breast tumors. Here, we investigated if diagnosis of triple-negative breast cancer (TNBC) independently increases risk of carrying a BRCA1/2 mutation in Pakistan. Methods Five hundred and twenty-three breast cancer patients including 237 diagnosed ≤ 30 years of age and 286 with a family history of breast/ovarian cancer were screened for BRCA1/2 small-range mutations and large genomic rearrangements. Immunohistochemical analyses were performed at one center. Univariate and multiple logistic regression models were used to investigate possible differences in prevalence of BRCA1/2 mutations according to patient and tumor characteristics. Results Thirty-seven percent of patients presented with TNBC. The prevalence of BRCA1 mutations was higher in patients with TNBC than non-TNBC (37 % vs. 10 %, P

Background Hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome (LS) account for a significant proportion of inherited gynecologic malignancies, mainly caused by pathogenic germline mutations in the BRCA1 and BRCA2 genes or in mismatch repair (MMR) genes, such as MLH1 and MSH2. Women harboring deleterious mutations in these genes have increased life-time risks of developing a number of malignancies including ovarian cancer. Since there is a phenotypic overlap of HBOC and LS, timely identification of individuals at-risk of a particular syndrome is crucial in order to optimize cancer risk management. Case presentation We report a novel pathogenic MSH2 mutation, c.2656G > T, which was identified in a 67-year-old female patient with breast cancer, who had previously tested negative for a deleterious mutation in the breast cancer susceptibility genes BRCA1, BRCA2, CHEK2 or RAD51C. The patient reported a personal history of endometrial cancer diagnosed at age 48, and a strong family history of breast and ovarian cancer, as well as several other malignancies within the spectrum of LS. The novel mutation was also found in the index patient’s daughter and a niece, who were diagnosed with endometrial and ovarian cancer, respectively. Breast and endometrial tumors from c.2656G > T mutation carriers showed loss of MSH2 and MSH6 protein expression. The mutation was absent in the control population. Conclusions Our finding suggests that testing for MMR genes may be of benefit to BRCA1/2 negative families with overlapping HBOC and LS phenotype in Pakistan. It is clinically significant to identify individuals harboring mutations in genes linked with a particular syndrome so that they can benefit from targeted life-saving cancer surveillance and preventive strategies.

Pathogenic germ line mutations in the BRCA1 and BRCA2 genes confer elevated risk of breast and ovarian cancer in females and have a link with variety of cancers other than breast cancer in males. Here we report the first case of a 45-year-old Pakistani male with renal cell carcinoma who was identified to harbor a disease-associated BRCA1 germ line mutation, 2080insA. Detailed description of the family history, clinical presentation and histopathological features of the renal cell carcinoma are presented.

In South America, a high proportion of the population is of Hispanic origin with an important representation in Colombia. Since nothing is known about the contribution of BRCA1 and BRCA2 germline mutations to hereditary breast/ovarian cancer in the Hispanic population from Colombia, we conducted the first study of 53 breast/ovarian cancer families from this country. Comprehensive BRCA mutation screening was performed using a range of techniques, including DHPLC, SSCP, and PTT, followed by DNA sequencing analysis. Thirteen deleterious germline mutations (24.5%) were identified in 53 families, comprising eight in BRCA1 and five in BRCA2. The two recurrent BRCA1 mutations, 3450 delCAAG and A1708E, accounted for 100% of all BRCA1 mutations identified in this cohort and the recurrent 3034 delACAA BRCA2 mutation for 40% of all BRCA2 mutations. Haplotype analyses suggested that each of these mutations has arisen from a common ancestor. The prevalence of BRCA1 or BRCA2 mutations was 50% in multiple case breast cancer families, and was 33% for the breast-ovarian cancer families. Our findings show that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Colombia. The spectrum of mutations differed completely to that previously reported in Hispanic families of predominantly Mexican origin from Southern California [1] suggesting that specific genetic risk assessment strategies for the different Hispanic populations in South America and in the United States need to be developed.

To the editor About 80% of breast and ovarian cancer families and about 50% of site-specific breast cancer families are due to germ line mutations in either BRCA1 (MIM 113705) or BRCA2 (MIM 600185) [1–3]. Women carrying a pathogenic mutation in BRCA1 or BRCA2 face average lifetime risks of 65% and 45%, respectively, of developing breast cancer, and corresponding ovarian cancer risks of 39% and 11% [4]. In humans, almost all BRCA carriers have been found to be heterozygous [5, 6]. The lack of viable homozygous BRCA mutation carriers in humans suggests that homozygous fetuses may be more susceptible to spontaneous abortions. Further evidence for this hypothesis comes from in vivo animal studies showing that mice homozygous for mutations in the BRCA1 or BRCA2 gene die early in embryogenesis [7, 8]. Two recent studies have explored the impact of BRCA mutation status on the occurrence of miscarriages. One study included BRCA carriers of Ashkenazi Jewish descent [9], and the other included mutation carriers from United States and Canada [10]. Neither study found a significant difference in the rate of miscarriage between BRCA carriers and non-carriers. We conjectured that miscarriages may occur more frequently in BRCA carriers from consanguineous parents since their offspring are more often homozygous by descent compared to those of unrelated parents, and living homozygous BRCA mutation carriers have not been observed. To explore this question, we studied the rates of miscarriages in Pakistani BRCA mutation carriers and non-carriers with a history of parental consanguinity. Pakistan has one of the highest rates of consanguinity in the world (range 60–70%) [11, 12]. Breast and/or ovarian cancer families were recruited at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, between June 2001 and November 2004. Of the 165 female index patients, 30 carried a deleterious BRCA mutation, and 135 were without any identifiable mutation. All families, screening methods and mutations have been described previously [13]. Four additional families carrying a deleterious BRCA mutation identified recently were also included in this study. Relevant information regarding potential breast cancer risk factors including family history of cancer, hormone use, reproductive history, medical history and life style factors was collected by interview-administered questionnaires. Detailed information on the outcome of each pregnancy (year of each pregnancy, number of miscarriages including spontaneous and therapeutic abortions, number of still and live born), and parental consanguinity was collected. Spontaneous abortion was defined as loss of a pregnancy before the completion of 20 gestational weeks from the M. U. Rashid F. Rasheed F. Sultan Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan

Breast cancer is the most common cancer among women worldwide. Approximately 5–10 % of breast cancers are hereditary, and caused by monoallelic germ line mutations in high-, moderate-, and low-penetrance breast cancer susceptibility genes. Several of these genes such as BRCA1, BRCA2, PALB2, BRIP1, and genes of the RAD51 family play an important role in maintenance of genomic stability and are functionally linked with homologous recombination-mediated DNA damage repair. Breast cancer susceptibility is connected with the Fanconi anemia (FA) pathway, since biallelic mutations in at least four genes, BRCA2 (FANCD1) [1], PALB2 (FANCN) [2], BRIP1 (FANCJ) [3], and RAD51C (FANCO) [4] have been shown to also cause FA. Recently, Kiiski and colleagues identified the FA complementation group M (FANCM) gene as a novel breast cancer susceptibility gene [5]. A nonsense mutation in exon 20, c.5101C[T (p.Q1701X), was identified by exome sequencing of germline DNA samples from 24 breast cancer patients from eleven BRCA1/2-negative Finnish breast cancer families. Further genotyping of a large sample set of breast or ovarian cancer cases and controls revealed a 3.5-fold increased frequency among triple-negative breast cancer (TNBC) cases and an approximately twofold increased mutation frequency among all breast cancer cases compared with controls. Given that deleterious mutations in other FA genes including FANCD1 (BRCA2) and FANCO (RAD51C) have previously been identified among TNBC patients from Pakistan [6, 7], we investigated whether the FANCM c.5101C[T mutation is associated with TNBC in Pakistan. Constitutional genomic DNA of 117 BRCA1/2-negative TNBC patients and 188 controls from Pakistan were screened for the FANCM c.5101C[T mutation. Of the TNBC cases, 75 were diagnosed with early-onset disease (B30 years of age), 37 belonged to families with C2 breast cancer cases, and five to breast–ovarian cancer families (Table 1). The median age of diagnosis was 28 years (range 18–67). Forty-four of the cases have been previously described [7]. Screening for the c.5101C[T mutation was performed using denaturing high-performance liquid chromatography (DHPLC) analyses followed by DNA sequencing of variant fragments. PCR primer sequences were as previously described [5]. DHPLC running conditions are available upon request. A positive mutation control was included in all DHPLC runs. The c.5101C[T mutation was not identified in any of the 117 TNBC cases and 188 controls implying that this mutation does not or rarely contributes to TNBC in this population. In line with our data, this mutation was also not detected in previous studies among 965 Icelandic unselected breast cancer patients including 92 BRCA1/2negative familial cases [5], 95 BRCA1/2-negative familial cases from Spain [8], and 3409 BRCA1/2-negative familial cases and 3896 controls from Italy, Netherland, Australia, and Spain [9]. The findings of these studies suggest that the c.5101C[T may be specific to the Finnish population. In the current study, we identified two other FANCM missense mutations. A mutation, c.4931G[A (p.R1644Q), & Muhammad U. Rashid usmanr@skm.org.pk