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1.
Crit Rev Food Sci Nutr ; 60(3): 351-374, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30614244

RESUMO

Brain is a central and pivotal organ of human body containing the highest lipids content next to adipose tissue. It works as a monitor for the whole body and needs an adequate supply of energy to maintain its physiological activities. This high demand of energy in the brain is chiefly maintained by the lipids along with its reservoirs. Thus, the lipid metabolism is also an important for the proper development and function of the brain. Being a prominent part of the brain, lipids play a vast number of physiological activities within the brain starting from the structural development, impulse conduction, insulation, neurogenesis, synaptogenesis, myelin sheath formation and finally to act as the signaling molecules. Interestingly, lipids bilayer also maintains the structural integrity for the physiological functions of protein. Thus, in light to all of these activities, lipids and its metabolism can be attributed pivotal for brain health and its activities. Decisively, the impaired/altered metabolism of lipids and its intermediates puts forward a key step in the progression of different brain ailments including neurodegenerative, neurological and neuropsychiatry disorders. Depending on their associated underlying pathways, they serve as the potential biomarkers of these disorders and are considered as necessary diagnostic tools. The present review discusses the role and level of altered lipids metabolism in brain diseases including neurodegenerative diseases, neurological diseases, and neuropsychiatric diseases. Moreover, the possible mechanisms of altered level of lipids and their metabolites have also been discussed in detail.

2.
J Pak Med Assoc ; 69(12): 1903-1906, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31853126

RESUMO

Intellectual disability (ID) or Mental Retardation (MR) is a broad term, which occupies several medical directions. It is extremely heterogeneous and has about reported 25,000 genes of which half of the genes expression have been found in the brain. Intellectual disability causes severe disability and has a worldwide prevalence of around 2% while autosomal recessive form of ID causes almost 25% of all non syndromic (NS) ID cases. A consanguineous family (who will be referred as) MR7 with phenotype of ID was sampled in Swat region of Pakistan. All affected individuals in the family were observed having a low IQ and cognitive mutilation with no sign of biochemical, skeletal or neurological abnormalities. Their dc-ribonucleic acid (DNA) was extracted and subjected to STS (Single tagged sequence) marker analyses which showed exclusion of all known non syndromic autosomal recessive (NS-AR) ID genes. In the family MR7, autozygosity mapping was performed by microarray single-nucleotide polymorphism analysis in all the collected samples, for a close examination of the homozygous region in all the affected however no homozygosity was observed for the normal parent. In this consanguineous family of Pakistan, autozygosity mapping showed linkage interval (chr14: 30,294,526- 32,106,658) overlapping with already reported MRT9 locus (chr14:26,578,608-32,780,288) for NS- ARID.

3.
J Clin Neurosci ; 67: 19-23, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31281085

RESUMO

Hereditary Spastic paraplegias (HSPs) are heterogeneous group of degenerative disorders characterized by progressive weakness and spasticity of the lower limbs, combined with additional neurological features. This study aimed to identify causative gene variants in two nonrelated consanguineous Pakistani families segregating HSP. Whole exome sequencing (WES) was performed on a total of five individuals from two families including four affected and one phenotypically normal individual. The variants were validated by Sanger sequencing and segregation analysis. In family A, a novel homozygous variant c.604G > A (p.Glu202Lys) was identified in the CYP2U1 gene with clinical symptoms of SPG56 in 3 siblings. Whereas, a previously reported variant c.5769delT (p.Ser1923Argfs*28) in the SPG11 gene was identified in family B manifesting clinical features of SPG11 in 3 affected individuals. Our combined findings add to the clinical and genetic variability associated with CYP2U1 and SPG11 variants highlighting the complexity of HSPs. These findings further emphasize the usefulness of WES as a powerful diagnostic tool.


Assuntos
Família 2 do Citocromo P450/genética , Proteínas/genética , Paraplegia Espástica Hereditária/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Criança , Feminino , Humanos , Masculino , Mutação , Paquistão , Linhagem , Fenótipo
4.
Int J Neurosci ; 129(9): 890-895, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30892110

RESUMO

Aim: Neuronal ceroid lipofuscinosis (NCLs) are the most common neurodegenerative disorders, with global incidence of 1 in 100,000 live births. NCLs affect central nervous system, primarily cerebellar and cerebral cortices. Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is the most common form of NCLs. JNCL is primarily caused by pathogenic mutations in CLN3 gene, which encodes a transporter transmembrane protein of uncertain function. The 1.02 kb deletion is the most common mutation in CLN3 that results in frame shift and a premature termination leading to nonfunctional protein. Here, we invetigated a large consanguineous family consisting of four affected individuals with clincal symptoms suggestive of Juvenile neuronal ceroid lipofuscinosis. Materials and methods: We conducted clinial and radilogical investigation of the family and performed NGS based Gene Panel sequencing comprising of five hundred and forty five candidate genes to characterize it at genetic level. Results: We identified a novel homozygous c.181_183delGAC mutation in the CLN3 gene seggregating witht the disorder in the family. The mutation induces in-frame deletion, deleting one amino acid (p.Asp61del) in CLN3 protein. The deleted amino acid aspartic acid plays an important role as general acid in enzymes active centers as well as in maintaining the ionic character of proteins. Conclusion: Our finding adds to genetic variability of Juvenile neuronal ceroid lipofuscinosis associated with CLN3 gene and a predicted CLN3 protein interacting domain site.


Assuntos
Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Mutação/genética , Lipofuscinoses Ceroides Neuronais/diagnóstico por imagem , Lipofuscinoses Ceroides Neuronais/genética , Adolescente , Humanos , Masculino , Paquistão , Linhagem
5.
Cancer Manag Res ; 11: 1087-1096, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30774437

RESUMO

Background: Delay in seeking health care by breast cancer patients is associated with advanced stage of disease at presentation and poor survival rates. This study aimed to identify the reasons for delayed presentation and their association with various sociodemographic variables. Methods: A total of 200 female patients with abnormal clinical findings, ie, lump or palpable mass, were consecutively invited for this study. Diagnostic delay was defined as a consultation with a health care provider more than 3 months from the appearance of the first symptoms. Sociodemographic variables, presenting symptoms, knowledge about diseases and its symptoms, time between seeking medical attention after appearance of symptoms and causes of delayed presentation were investigated. Chi-squared and logistic regression tests for significance and associations were used. Results: Among 125 women with breast cancer fulfilling the inclusion criteria, aged 24-75 years, 88.8% (n=111) presented late (≥3 months) and 59% presented with advanced stage of disease (stage III/IV). The majority (65.6%) were older than 40 years of age, 99.2% were married, 60.8% had <8 years of education, 67.2% had poor social status, and 64.8% had a negative family history of any cancer type. Almost all patients (96%) complained about the presence of a painless lump in their breast. Ignorance of disease or the presence of painless lumps in the breast and low financial resources for therapy (81.1%) were the main variables associated with delayed presentation. Educational factors (P<0.001, OR 4.682) and social status (P<0.001, OR 1.8) were also associated with delayed presentation. Conclusion: Our study highlighted the variables associated with delayed presentation in Pakistani breast cancer patients. A significant number of patients presented late owing to misconceptions and poor knowledge about the disease and its symptoms, while illiteracy and poor social status were the major contributing factors for delayed presentation, resulting in an advanced presentation of disease and ultimately a decreased survival rate.

6.
Lipids Health Dis ; 18(1): 26, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683111

RESUMO

Brain is a vital organ of the human body which performs very important functions such as analysis, processing, coordination, and execution of electrical signals. For this purpose, it depends on a complex network of nerves which are ensheathed in lipids tailored myelin; an abundant source of lipids in the body. The nervous system is enriched with important classes of lipids; sphingolipids and cholesterol which compose the major portion of the brain particularly in the form of myelin. Both cholesterol and sphingolipids are embedded in the microdomains of membrane rafts and are functional units of the neuronal cell membrane. These molecules serve as the signaling molecules; hold important roles in the neuronal differentiation, synaptogenesis, and many others. Thus, their adequate provision and active metabolism are of crucial importance in the maintenance of physiological functions of brain and body of an individual. In the present review, we have highlighted the physiological roles of cholesterol and sphingolipids in the development of the nervous system as well as the association of their altered metabolism to neurological and neurodegenerative diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Colesterol/metabolismo , Doenças do Sistema Nervoso/genética , Esfingolipídeos/metabolismo , Animais , Encéfalo/metabolismo , Membrana Celular/genética , Colesterol/genética , Humanos , Lipídeos/genética , Microdomínios da Membrana/genética , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Esfingolipídeos/genética
7.
Fam Cancer ; 18(2): 261-265, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30478739

RESUMO

Precise genetic counseling and prenatal diagnosis are often hindered by incomplete penetrance of risk variance and complex patterns of inheritance. Here, we performed a clinical and genetic study of a five-generation Pakistani family with a history of multiple cases of childhood brain tumors. Six affected individuals died of brain tumors at very early ages and three were confirmed as having a homozygous mutation in exon 6 of the PMS2 gene (c.543delT). Fifteen members of the family were identified as heterozygous carriers of this mutation with a lack of cancer incidence. Both clinical manifestations and genetic test results of brain tumor patients in the family support the diagnosis of constitutional mismatch repair deficiency (CMMRD) syndrome, a condition in which individuals carry homozygous germline mutations in mismatch repair machinery genes with an early onset of malignancies such as glioma. This information was used to guide prenatal diagnosis with genetic testing on chorionic villus samples for the family. This is the first report of prenatal genetic diagnosis of hereditary brain tumor.


Assuntos
Neoplasias Encefálicas/diagnóstico , Amostra da Vilosidade Coriônica , Neoplasias Colorretais/diagnóstico , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Síndromes Neoplásicas Hereditárias/diagnóstico , Fatores Etários , Idade de Início , Neoplasias Encefálicas/genética , Criança , Pré-Escolar , Neoplasias Colorretais/genética , Consanguinidade , Éxons/genética , Feminino , Aconselhamento Genético , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Síndromes Neoplásicas Hereditárias/genética , Paquistão , Linhagem , Gravidez
8.
Bioconjug Chem ; 30(1): 13-28, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30508381

RESUMO

For SLN lymph node biopsy (SLNB), SLN mapping has become a standard of care procedure that can accurately locate the micrometastases disseminated from primary tumor sites to the regional lymph nodes. The broad array of SLN mapping has prompted the development of a wide range of SLN tracers, rationally designed for noninvasive and high-resolution imaging of SLNs. At present, conventional SLN imaging probes (blue dyes, radiocolloids, and few other small-molecular dyes), although serving the clinical needs, are often associated with major issues such as insufficient accumulation in SLN, short retention time, staining of the surgical field, and other adverse side effects. In a recent advancement, newly designed fluorescent nanoprobes are equipped with novel features that could be of high interest in SLN mapping such as (i) a unique niche that is not met by any other conventional SLN probes, (ii) their adoptable synthesis method, and (ii) excellent sensitivity facilitating high resolution SLN mapping. Most importantly, lots of effort has been devoted for translating the fluorescent nanoprobes into a clinical setup and also imparting the multimodal imaging abilities of nanoprobes for the excellent diagnosis of life-threatening diseases such as cancer. In this review, we will provide a detailed roadmap of the progress of a wide variety of current fluorescent molecular probes and emphasize the future of nanomaterial-based single/multimodal imaging probes that have true potential translation abilities for SLN mapping.


Assuntos
Corantes Fluorescentes/química , Sondas Moleculares/química , Biópsia de Linfonodo Sentinela/métodos , Animais , Humanos , Verde de Indocianina/química , Metástase Linfática , Micrometástase de Neoplasia , Teoria Quântica , Linfonodo Sentinela/patologia , Solubilidade
9.
J Clin Invest ; 128(10): 4313-4328, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30179222

RESUMO

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.


Assuntos
Síndrome Nefrótica/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Xenopus/genética , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
10.
Genes Genomics ; 40(5): 553-559, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29892959

RESUMO

Retinitis pigmentosa (RP) is the most frequent genetically and clinically heterogeneous inherited retinal degeneration. To date, more than 80 genes have been identified that cause autosomal dominant, autosomal recessive and X linked RP. However, locus and allelic heterogeneity of RP has not been fully captured yet. This heterogeneity and lack of an accurate genotype phenotype correlation makes molecular dissection of the disease more difficult. The present study was designed to characterize the underlying pathogenic variants of RP in Pakistan. For this purpose, a large consanguineous family with RP phenotype showing autosomal recessive mode of inheritance was selected after a complete ophthalmological examination. Next generation sequencing was used for the identification of molecular determinant followed by Sanger-sequencing for confirmation. After sequence analysis a novel homozygous missense mutation, (c.602 C > T) in exon 4 of the RDH5 gene (MIM: 601617) was identified. This mutation resulted in substitution of phenyl alanine for serine at amino acid 201 (p.Ser201Phe) of the RDH5 gene. The same mutation was not detected in the 200 ethnically-matched control samples by Sanger sequencing. The identified mutant allele segregated in homozygous fashion in all the affected individuals of pedigree. Identification of this mutation reveals the allelic heterogeneity of RDH5 in patients with RP phenotype. The findings of this study demonstrate the clinical significance of next generation sequencing to understand the molecular basis of diseases and would help to reveal new proteins and their function in visual cycle will pave the way for early diagnosis, genetic counseling and better therapeutic inventions.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/fisiologia , Retinite Pigmentosa/genética , Adolescente , Adulto , Alelos , Consanguinidade , Proteínas do Olho/genética , Família , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Mutação de Sentido Incorreto/genética , Paquistão , Linhagem , Polimorfismo de Nucleotídeo Único/genética
11.
Ann Neurol ; 82(4): 562-577, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28892560

RESUMO

OBJECTIVE: Autosomal recessive primary microcephaly (MCPH) is a rare condition characterized by a reduced cerebral cortex accompanied with intellectual disability. Mutations in 17 genes have been shown to cause this phenotype. Recently, mutations in CIT, encoding CRIK (citron rho-interacting kinase)-a component of the central spindle matrix-were added. We aimed at identifying novel MCPH-associated genes and exploring their functional role in pathogenesis. METHODS: Linkage analysis and whole exome sequencing were performed in consanguineous and nonconsanguineous MCPH families to identify disease-causing variants. Functional consequences were investigated by RNA studies and on the cellular level using immunofluorescence and microscopy. RESULTS: We identified homozygous mutations in KIF14 (NM_014875.2;c.263T>A;pLeu88*, c.2480_2482delTTG; p.Val827del, and c.4071G>A;p.Gln1357=) as the likely cause in 3 MCPH families. Furthermore, in a patient presenting with a severe form of primary microcephaly and short stature, we identified compound heterozygous missense mutations in KIF14 (NM_014875.2;c.2545C>G;p.His849Asp and c.3662G>T;p.Gly1221Val). Three of the 5 identified mutations impaired splicing, and 2 resulted in a truncated protein. Intriguingly, Kif14 knockout mice also showed primary microcephaly. Human kinesin-like protein KIF14, a microtubule motor protein, localizes at the midbody to finalize cytokinesis by interacting with CRIK. We found impaired localization of both KIF14 and CRIK at the midbody in patient-derived fibroblasts. Furthermore, we observed a large number of binucleated and apoptotic cells-signs of failed cytokinesis that we also observed in experimentally KIF14-depleted cells. INTERPRETATION: Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577.


Assuntos
Citocinese/genética , Regulação da Expressão Gênica/genética , Cinesina/genética , Microcefalia/genética , Mutação/genética , Proteínas Oncogênicas/genética , Caspase 7/metabolismo , Movimento Celular/genética , Células Cultivadas , Criança , Pré-Escolar , Saúde da Família , Feminino , Fibroblastos/fisiologia , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Microcefalia/diagnóstico por imagem , Microcefalia/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Tubulina (Proteína)/metabolismo
12.
Eur J Hum Genet ; 25(7): 848-853, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28488678

RESUMO

Spinocerebellar ataxias (SCA) comprise a heterogeneous group of inherited neurological disorders characterized by a range of symptoms from both cerebellar and extra cerebellar structures. We investigated the cause of autosomal recessive, congenital SCA in six affected family members from a large consanguineous family. Using whole-exome sequencing, we identified a homozygous ITPR1 missense variant [c.5360T>C; p.(L1787P)] segregating in all affected individuals. Heterozygous carriers were asymptomatic despite cerebellar hypoplasia. Variants in the ITPTR1 gene have previously been associated exclusively with autosomal dominant SCA15 and SCA29 with slow or no progression. The L1787 residue is highly conserved and the leucine to proline substitution has a predicted destabilizing effect on the protein structure. Additionally, the L1787P variant is located in a domain separated from previously described and dominant-acting missense variants consistent with a distinct effect on IP3R1 tetramer structure and function. Taken together, we show for the first time that a biallelic ITPR1 missense variant may cause an autosomal recessive and infantile onset SCA29, albeit with subclinical cerebellar hypoplasia in carriers. Our findings add to the genetic complexity of SCA29 and broaden the correlations between ITPR1 variants and their clinical expression.


Assuntos
Cerebelo/anormalidades , Heterozigoto , Receptores de Inositol 1,4,5-Trifosfato/genética , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Homozigoto , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Masculino , Pessoa de Meia-Idade , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/patologia , Linhagem , Estabilidade Proteica
13.
Mol Genet Genomics ; 292(2): 365-383, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28004182

RESUMO

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/fisiologia , Proteínas de Ciclo Celular , Movimento Celular , Tamanho Celular , Células Cultivadas , Centrossomo/ultraestrutura , Códon sem Sentido , DNA/genética , Fibroblastos/metabolismo , Ligação Genética , Predisposição Genética para Doença , Genoma Humano , Células HEK293 , Células HeLa , Fator de Crescimento de Hepatócito/metabolismo , Homozigoto , Humanos , Mutação , Tamanho do Órgão , Linhagem , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo
14.
Hum Genet ; 135(2): 157-70, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26621532

RESUMO

Primary microcephaly is a disorder characterized by a small head and brain associated with impaired cognitive capabilities. Mutations in 13 different genes encoding centrosomal proteins and cell cycle regulators have been reported to cause the disease. CASC5, a gene encoding a protein important for kinetochore formation and proper chromosome segregation during mitosis, has been suggested to be associated with primary microcephaly-4 (MCPH4). This was based on one mutation only and circumstantial functional evidence. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a second mutation (NM_170589.4;c.6673-19T>A) in CASC5. This mutation induced skipping of exon 25 of CASC5 resulting in a frameshift and the introduction of a premature stop codon (p.Met2225Ilefs*7). The C-terminally truncated protein lacks 118 amino acids that encompass the region responsible for the interaction with the hMIS12 complex, which is essential for proper chromosome alignment and segregation. Furthermore, we showed a down-regulation of CASC5 mRNA and reduction of the amount of CASC5 protein by quantitative RT-PCR and western blot analysis, respectively. As a further sign of functional deficits, we observed dispersed dots of CASC5 immunoreactive material outside the metaphase plate of dividing patient fibroblasts. Normally, CASC5 is a component of the kinetochore of metaphase chromosomes. A higher mitotic index in patient cells indicated a mitotic arrest in the cells carrying the mutation. We also observed lobulated and fragmented nuclei as well as micronuclei in the patient cells. Moreover, we detected an altered DNA damage response with higher levels of γH2AX and 53BP1 in mutant as compared to control fibroblasts. Our findings substantiate the proposed role of CASC5 for primary microcephaly and suggest that it also might be relevant for genome stability.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Homozigoto , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Processamento de RNA , Sequência de Aminoácidos , Células Cultivadas , Segregação de Cromossomos , Códon sem Sentido/genética , Códon sem Sentido/metabolismo , Dano ao DNA/genética , Regulação para Baixo , Éxons , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Mutação da Fase de Leitura , Ligação Genética , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Cinetocoros/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Paquistão , Linhagem , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
16.
J Med Genet ; 52(9): 599-606, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26142438

RESUMO

BACKGROUND: Keratosis pilaris atrophicans (KPA) is a group of rare genodermatoses characterised by perifollicular keratosis and inflammation that progresses to atrophy and scars of the facial skin. Keratosis pilaris of extensor areas of limbs is a common associated finding. Most cases with KPA are sporadic and no consistent inheritance pattern has been documented. METHODS: A large consanguineous Pakistani pedigree segregating autosomal recessive KPA of a mixed type was subject to autozygosity mapping and whole exome sequencing. Quantification of mRNA and protein levels was performed on fibroblasts from affected individuals. Cellular uptake of the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) ligand α2-macroglobulin (α(2)M) was quantified using fluorescence confocal microscopy. RESULTS: Genetic analyses identified a unique homozygous missense variant (K1245R) in the LRP1 in all affected family members. LRP1 encodes the LRP1, a multifunctional cell surface receptor with endocytic functions that belongs to the LDL receptor family. The LRP1 mRNA and LRP1 protein levels in fibroblasts of affected individuals were markedly reduced when compared with controls. Similarly, the LRP1-mediated cellular uptake of α(2)M was reduced in patient fibroblasts. CONCLUSIONS: This is the first report on LRP1 as a pathogenic gene for autosomal recessive KPA and keratosis pilaris. The inflammatory characteristics of the KPA entity in our family suggest a link to the immune-regulatory functions of LRP1.


Assuntos
Anormalidades Múltiplas/genética , Doença de Darier/genética , Éxons , Sobrancelhas/anormalidades , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutação Puntual , Transtornos Cromossômicos/genética , Consanguinidade , Exoma , Genes Recessivos , Humanos , Paquistão , Linhagem , Análise de Sequência de DNA
17.
Artigo em Inglês | MEDLINE | ID: mdl-25969822

RESUMO

BACKGROUND: The Catechol-O-Methyl Transferase (COMT) gene polymorphism (I/D of C nucleotide at base position 900) has been previously implicated in the development of type 2 diabetes (T2D) and kidney disease. So, aim of this study was to find association of I/D polymorphism with T2D, and its associated factors like family history and nephropathy (End Stage Renal Disease, ESRD) patients in a cohort of Pakistani Punjabis. METHODS: Genomic DNA was extracted from human subjects divided as four study groups: controls (n = 46), diabetics (n = 46), diabetics with nephropathy/ESRD (n = 53), and non-diabetics without nephropathy/ESRD (n = 43). The 900 I/D C polymorphism in the COMT gene was tested by PCR-RFLP method. Genotype and allele frequencies as well as Odds Ratios were calculated for these groups. Groups were compared statistically for the analysis of genotypes, alleles, biochemical parameters as well as disease status. RESULTS: In comparison with control group (non-diabetic, non-nephropathy), there was no significant difference in rest of the three groups for allele or genotype frequencies of COMT gene. However, Chi square (χ(2)) analysis identified a significant (p = 0.02) correlation of the 900 I/D C polymorphism with family history of diabetes, as it was found that greater number (74%) of patients having I allele had a positive family history of T2D. CONCLUSIONS: A significant correlation of the COMT polymorphism (900 I/D C) with the family history of T2D has been observed, which has not been previously reported in Pakistani Punjabi population, however, this preliminary finding requires further validation studies.

18.
BMC Med Genet ; 15: 133, 2014 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-25496299

RESUMO

BACKGROUND: Cerebral palsy (CP) is a heterogeneous neurodevelopmental disorder associated with intellectual disability in one-third of cases. Recent findings support Mendelian inheritance in subgroups of patients with the disease. The purpose of this study was to identify a novel genetic cause of paraplegic CP with intellectual disability in a consanguineous Pakistani family. METHODS: We performed whole-exome sequencing (WES) in two brothers with CP and intellectual disability. Analysis of AP4M1 mRNA was performed using quantitative real-time PCR on total RNA from cultured fibroblasts. The brothers were investigated clinically and by MRI. RESULTS: We identified a novel homozygous AP4M1 mutation c.194_195delAT, p.Y65Ffs*50 in the affected brothers. Quantitative RT-PCR analysis showed markedly reduced AP4M1 mRNA levels suggesting partial non-sense mediated mRNA decay. Several clinical and MRI features were consistent with AP-4 complex deficiency. However, in contrast to previously reported cases with AP4M1 mutations our patients show an aggressive behavior and a relatively late onset of disease. CONCLUSION: This study shows an AP4M1 mutation associated with aggressive behavior in addition to mild dysmorphic features, intellectual disability, spastic paraparesis and reduced head circumference. Our findings expand the clinical spectrum associated with AP-4 complex deficiency and the study illustrates the importance of MRI and WES in the diagnosis of patients with CP and intellectual disability.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Paralisia Cerebral/genética , Mutação , Adolescente , Paralisia Cerebral/complicações , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Genes Recessivos , Homozigoto , Humanos , Lactente , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Imagem por Ressonância Magnética , Masculino , Linhagem , RNA Mensageiro/metabolismo
19.
Nat Genet ; 46(12): 1283-1292, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25344692

RESUMO

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.


Assuntos
Transtornos do Crescimento/genética , Microcefalia/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Degeneração Retiniana/genética , Adolescente , Adulto , Animais , Centríolos/ultraestrutura , Criança , Pré-Escolar , Saúde da Família , Feminino , Fibroblastos/metabolismo , Genótipo , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Repetições de Microssatélites , Proteínas Associadas aos Microtúbulos/genética , Mitose , Paquistão , Linhagem , Fenótipo , Adulto Jovem , Peixe-Zebra
20.
Mol Biol Rep ; 41(9): 5585-91, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25063576

RESUMO

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.


Assuntos
Análise Custo-Benefício , Diabetes Mellitus Tipo 2/genética , Técnicas de Genotipagem/métodos , PPAR gama/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , Sequência de Bases , Bioensaio , Estudos de Casos e Controles , Países em Desenvolvimento , Estudo de Associação Genômica Ampla , Humanos , Dados de Sequência Molecular , PPAR gama/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNA
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