novel mutation in SNX10 gene causes malignant infantile osteopetrosis

Background: Osteopetrosis is a group of genetically heterogonous diseases and the main feature of that is increased bone density due to osteoclast's abnormality. It has three clinical forms based on inheritance pattern, severity and age of onset: the dominant benign form [ADO], the intermediate form [IRO] and the recessive severe form [ARO]. One of the recently discovered genes for ARO form is SNX10 that accounts for 4% of affected persons by this type

Methods: In this paper, a 15 years old girl affected by osteopetrosis has been analyzed for detecting causal mutation in known osteopetrosis genes. To get it done, amplified exons of the genes were sequenced and then were analyzed

Results: Direct sequencing of SNX10 gene showed a homozygous c.43delG variant in the patient. Both healthy parents were heterozygous for this variant. In silico analysis revealed that this novel variant can be considered as the cause of disease in the patient

Conclusion: In this paper, a girl affected by osteopetrosis with a novel deletion in SNX10 gene was reported

novel variant in the PAH gene causing phenylketonuria in an Iranian pedigree

Background: Phenylalanine hydroxylase [PAH] gene is the well-known causative gene for classic Phenylketonuria [PKU] [OMIM#261600] disease, with more than 500 reported mutations. Through this study, a novel mutation in the PAH gene in an Iranian pedigree with phenylketonuria was introduced

Methods: A consanguineous family with a 10-year old affected girl was referred for genetic analysis. Mutation screening of all exons and exon-intron boundaries was performed by Sanger sequencing, and mini haplotype analysis was carried out by genotyping of Short Tandem Repeat [STR] and Variable Number Tandem Repeat [VNTR] alleles

Results: Mutation analysis revealed a novel homozygous insertion of a single adenine nucleotide at position 335 in exon 3 of the PAH gene. Based on the American College of Medical Genetics and Genomics [ACMG] guidelines, the change is interpreted as a pathogenic mutation which produces a premature termination signal [TAA] at codon 113 according to in silico assessments. The mini haplotype analysis showed that this mutation was linked to STR [15] -VNTR [3]

Conclusion: In this study, a novel mutation was reported in a patient who had PKU symptoms without any previously reported mutations in the PAH gene [NM_000277.1: p.Asp112Glufs*2] that can be responsible for the classical PKU phenotype in the Iranian population. Detection of novel mutations indicates notable allelic heterogeneity of the PAH locus among this population

Homozygosity Mapping and Targeted Sanger Sequencing Identifies Three Novel CRB1 [Crumbs homologue 1] Mutations in Iranian Retinal Degeneration Families

Background: Inherited retinal diseases [IRDs] are a group of genetic disorders with high degrees of clinical, genetic and allelic heterogeneity. IRDs generally show progressive retinal cell death resulting in gradual vision loss

IRDs constitute a broad spectrum of disorders including retinitis pigmentosa and Leber congenital amaurosis. In this study, we performed genotyping studies to identify the underlying mutations in three Iranian families

Methods: Having employed homozygosity mapping and Sanger sequencing, we identified the underlying mutations in the crumbs homologue 1 gene. The CRB1 protein is a part of a macromolecular complex with a vital role in retinal cell polarity, morphogenesis, and maintenance

Results: We identified a novel homozygous variant [c.1053-1061del; p.Gly352-Cys354del] in one family, a combination of a novel [c.2086T>C; p.Cys696Arg] and a known variant [c.2234C>T, p.Thr745Met] in another family and a homozygous novel variant [c.3090T>A; p.Asn!030Lys] in a third family

Conclusion: This study shows that mutations in CRB1 are relatively common in Iranian non-syndromic IRD patients

efficient trio-based mini-haplotyping method for genetic diagnosis of phenylketonuria

Objective: The phenylalanine hydroxylase [PAH] locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat [STR] genotyping

Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat [VNTR] were labeled with three different non-overlapping dyes 5-carboxyfluorescein [FAM], 6-carboxy-N,N,N',N'-tetramethylrhodamine [HEX] and 6-carboxy-N,N,N',N'-tetramethylrhodamine [TAMRA]. The polymerase chain reaction [PCR] products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software

Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift [migrated at a slower electrophoretic rate] relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability [P<0.001]

Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems

Functional analysis of a novel splicing mutation in the mutase gene of two unrelated pedigrees

Objective: methylmalonic acidura [MMA] is a rare autosomal recessive inborn error of metabolism. In this study we present a novel nucleotide change in the mutase [MUT] gene of two unrelated Iranian pedigrees and introduce the methods used for its functional analysis

Materials and Methods: two probands with definite diagnosis of MMA and a common novel variant in the MUT were included in a descriptive study. Bioinformatic prediction of the splicing variant was done with different prediction servers. Reverse transcription- polymerase chain reaction [RT-PCR] was done for splicing analysis and the products were analyzed by sequencing

Results: the included index patients showed elevated levels of propionylcarnitine [C3]. Urine organic acid analysis confirmed the diagnosis of MMA, and screening for mutations in the MUT revealed a novel C to G variation at the 3' splice acceptor site in intron 12. In silico analysis suggested the change as a mutation in a conserved sequence. The splicing analysis showed that the C to G nucleotide change at position -3 in the acceptor splice site can lead to retention of the intron 12 sequence

Conclusion: this is the first report of a mutation at the position -3 in the MUT intron 12 [c.2125-3C>G]. The results suggest that the identified variation can be associated with the typical clinical manifestations of MMA

Whole exome sequencing reveals a BSCL2 mutation causing progressive encephalopathy with lipodystrophy [PELD] in an Iranian pediatric patient

Background: Progressive encephalopathy with or without lipodystrophy is a rare autosomal recessive childhoodonset seipin-associated neurodegenerative syndrome, leading to developmental regression of motor and cognitive skills. In this study, we introduce a patient with developmental regression and autism. The causative mutation was found by exome sequencing

Methods: The proband showed a generalized hypertonia and regression of all developmental milestones. Based on the advantages of next-generation sequencing [NGS], whole exome sequencing [WES] was requested. The functional significance of variants was evaluated by NGS-specific prediction servers. Sanger sequencing was used for segregation analysis in the family

Results: There was no specific sign in the clinical and paraclinical investigations of the patient to establish a conclusive clinical diagnosis. WES detected a known homozygous nonsense mutation in BSCL2 [NM_001122955.3:c.985C>T; p.Arg329*]. The variant is segregating in the pedigree with an autosomal recessive pattern

Conclusion: Exome sequencing is a robust method for identifying the candidate gene variants in Mendelian traits

frequency of human leukocyte antigen-DRB1 alleles, using sequence-based genotyping in 68 parents-child trios study in Iranian subjects

Background: The human leukocyte antigen-DRB1 [HLA-DRB1] locus is one of the most polymorphic human loci and has a crucial role in the immune system. Assessing the allelic frequencies of HLA-DRB1 locus would be a fundamental factor in defining the origin of populations, relationships with other populations, disease association studies and the constitution of unrelated bone marrow donor registries. In the current study HLA-DRB1 alleles and their frequencies are determined in a family-based study by DNA sequencing-based typing high-resolution [2 field] level of typing

Materials and Methods: Genomic DNA from 3 members of 68 unrelated families [a total of 204 individuals] was extracted. Exon 2 of DRB1 gene was amplified and sequenced and allele assignment was performed using Assign SBT v4.7sequence analysis software

Results: We had DRB1*11:04 with frequency of 0.0931, DRB1*03:01 with 0.0882, DRB1*11:01 with 0.0735, DRB1*13:01 with 0.071 and also alleles DRB1*08:03, DRB1*13:42, DRB1*14:04 and DRB1*14:07 with frequency of 0.0024

Conclusion: A total of 34 different alleles were found in the study subjects with DRB1*11:04, DRB1*03:01, DRB1*11:01 being the most frequent alleles respectively

PKD2 mutation in an Iranian autosomal dominant polycystic kidney disease family with misleading linkage analysis data

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder caused by mutation in 2 genes PKD1 and PKD2. Thus far, no mutation is identified in approximately 10% of ADPKD families, which can suggest further locus heterogeneity. Owing to the complexity of direct mutation detection, linkage analysis can initially identify the responsible gene in appropriate affected families. Here, we evaluated an Iranian ADPKD family apparently unlinked to both PKD1 and PKD2 genes. This is one of the pioneer studies in genetic analysis of ADPKD in Iranian population. METHODS: Linkage reanalysis was performed by regenotyping of flanking microsatellite markers in 8 individuals of the ADPKD family. Direct mutation analysis was performed by Sanger sequencing. RESULTS: Mutation analysis revealed a pathogenic mutation (c.1094+1G>A) in the PKD2 gene in the proband. Analyzing 2 healthy and 4 clinically affected members confirmed the correct segregation of the mutation within the family and also ruled out the disease in 1 suspected individual. Misinterpretation of the linkage data was due to the occurrence of 1 crossing over between the PKD2 intragenic and the nearest downstream marker (D4S2929). Homozygosity of upstream markers caused the recombination indistinguishable. CONCLUSION: Although analysis of additive informative polymorphic markers can overcome the misleading haplotype data, it is limited because of the lack of other highly polymorphic microsatellite markers closer to the gene. Direct mutation screening can identify the causative mutation in the apparently unlinked pedigree; moreover, it is the only approach to achieve the confirmed diagnosis in individuals with equivocal imaging results.

Effect of embryonic cerebrospinal fluid on proliferation and differentiation of neuroprogenitor cells

Embryonic cerebrospinal fluid [e-CSF] has an important role in development of embryonic and adult brain. Proteomic analysis suggests that this fluid has many morphogenes and cytokines that alter in time and space throughout embryonic life. The aim of this study was to evaluate the developmental effect of embryonic CSF on proliferation and differentiation of neuroprogenitor cells in different gestational age. In this experimental study, we examined the role of e-CSF on proliferation and differentiation of neuroprogenitor cells using neurosphere culture method. Neurospheres size analysis and MTT assay were used to assess cell proliferation after four days in vitro. Glial differentiation study was carried out by immunocytochemistry. Neurospheres size and percentage of glial fibrialy acidic protein [GFAP] positive cells were measured by image analyzer [image J]. The data were analyzed by one-way ANOVA, followed by the Tukey's post hoc test. Data were expressed as mean +/- SEM, and differences were considered significant when p<0.05, 0.01 and 0.001. Viability and proliferation of neuro progenitor cells in cultures conditioned with E16 CSF and E18 CSF were significantly increased compare to control group. A dramatic decrease in percentage of GFAP-positive cells was found following the application of CSF from E16 and E18 embryos, but not E20 CSF. Our data suggest that, e-CSF altered proliferation and differentiation of neuro progenitor cells in age dependent manner. E16 and E18 CSF enhanced proliferation and viability of neuro progenitor cells, and inhibited differentiation to glial fate in comparison with control group

Producing a mammalian GFP expression vector containing neomycin resistance Gene

The green fluorescent protein [GFP] was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast, fungal plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded the 2008 noble prize in chemistry for their discovery and development of GFP. In many studies on mammalian cells, GFP gene is introduced into cells using vector-based systems or a recombinant virus to track the location of a target protein or to study the expression level of the gene of interest, but in these studies there is no selection marker to normalize transfection. According to the importance of neomycin gene as a selection marker in mammalian cells, we aimed to produce a GFP expression vector that contains neomycin gene. GFP gene was separated from pEGFP-Nl vector and was inserted in the backbone of pCDNA3.1/His/LacZ vector that contained the neomycin gene. The resulted vector contained GFP beside neomycin gene

introduction to RNA interference [RNAi]

The discovery of RNA interference [RNAi] in recent years has revolutionised biological and medical research. RNAi is the sequence-specific gene silencing triggered by double-stranded RNA [dsRNA]. Until a couple of years ago only biologists working with Caenorhabditis elegans and plants could use RNAi technology to gain insight into gene function. Tuschl and colleagues in 2001 showed that RNAi could also be used in mammalian cells to effectively inhibit endogenous gene expression in a sequence-specific manner. This remarkable work created high hopes of using RNAi as a potential therapy for many human diseases. Currently RNAi is widely used for functional genomics and for specific therapeutic intervention in preclinical models of different human diseases characterised by aberrant gene expression. This review provides an overview to the development of RNAi technology and its current applications in medicine