High‐resolution array‐CGH in patients with oculocutaneous albinism identifies new deletions of the TYR,OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene

Oculocutaneous albinism (OCA) is caused by mutations in six different genes, and their molecular diagnosis encompasses the search for point mutations and intragenic rearrangements. Here, we used high‐resolution array‐comparative genome hybridization (CGH) to search for rearrangements across exons, introns and regulatory sequences of four OCA genes: TYR, OCA2, TYRP1, and SLC45A2. We identified a total of ten new deletions in TYR, OCA2, and SLC45A2. A complex rearrangement of OCA2 was found in two unrelated patients. Whole‐genome sequencing showed deletion of a 184‐kb fragment (identical to a deletion previously found in Polish patients), whereby a large portion of the deleted sequence was re‐inserted after severe reshuffling into intron 1 of OCA2. The high‐resolution array‐CGH presented here is a powerful tool to detect gene rearrangements. Finally, we review all known deletions of the OCA1–4 genes reported so far in the literature and show that deletions or duplications account for 5.6% of all mutations identified in the OCA1–4 genes.     

Identification and characterization of the compound heterozygous variants of TYR gene in a northern Chinese family with Oculocutaneous albinism type 1

Oculocutaneous albinism (OCA) is a genetically heterogeneous disease and is most inherited in an autosomal recessive manner. The characteristic manifestation of OCA is due to disfunction of melanin synthesis. OCA1 is the most severe subtype of OCA and is caused by homozygous or compound heterozygous variants in tyrosinase (TYR) gene, which is the key gene for melanin synthesis. This study aimed to identify the genetic variants of a northern Chinese family with OCA1. Clinical information and peripheral blood samples were collected. PCR amplification and Sanger sequencing were used to detect the entire exons and adjacent flanking sequences of TYR gene. Functional prediction of variants was performed by various bioinformatic analyses, while the pathogenicity classification of variants was evaluated according to ACMG standards and guidelines. A missense variant NM_000372.5:c.107G > C;NP_000363.1:p.C36S was discovered in TYR gene which converted cysteine to serine. Another variant in intron, NM_000372.5:c.1037–7 T > A, also affected the function of TYR gene. We verified the pathogenicity of the intron variant with a pCAS2 mini-gene based splicing assay and found that c.1037–7 T > A led to an insertion of 5 bp upstream from the common acceptor site of exon 3, which caused a frameshift TYR:c.1037–7 T > A:p.G346Efs*11. The results showed that the compound heterozygous variants c.107G > C:p.C36S and c.1037–7 T > A:p.G346Efs*11 of TYR gene were the pathogenic variants for this OCA1 family.     

A Simple Definition of Structural Regions in Proteins and Its Use in Analyzing Interface Evolution

Analysis of proteins commonly requires the partition of their structure into regions such as the surface, interior, or interface. Despite the frequent use of such categorization, no consensus definition seems to exist. This study thus aims at providing a definition that is general, is simple to implement, and yields new biological insights. This analysis relies on 397, 196, and 701 protein structures from Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens, respectively, and the conclusions are consistent across all three species. A threshold of 25% relative accessible surface area best segregates amino acids at the interior and at the surface. This value is further used to extend the core-rim model of protein-protein interfaces and to introduce a third region called support. Interface core, rim, and support regions contain similar numbers of residues on average, but core residues contribute over two-thirds of the contact surface. The amino acid composition of each region remains similar across different organisms and interface types. The interface core composition is intermediate between the surface and the interior, but the compositions of the support and the rim are virtually identical with those of the interior and the surface, respectively. The support and rim could thus “preexist” in proteins, and evolving a new interaction could require mutations to form an interface core only. Using the interface regions defined, it is shown through simulations that only two substitutions are necessary to shift the average composition of a  1000-Ų surface patch involving ∼ 28 residues to that of an equivalent interface. This analysis and conclusions will help understand the notion of promiscuity in protein-protein interaction networks.     

In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants

Tyrosinase related protein 1 (TYRP1) is the most abundant melanosomal protein of the melanocyte, where plays an important role in the synthesis of eumelanin, possibly catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic acid. Mutations to the TYRP1 gene can result in oculocutaneous albinism type 3 (OCA3), a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn²⁺ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial protein misfolding as the possible mechanism of protein malfunction caused by OCA3 inherited mutations.     

Mutational analysis of FUS gene and its structural and functional role in amyotrophic lateral sclerosis 6

Amyotrophic lateral sclerosis 6 (ALS6) is an autosomal recessive disorder caused by heterozygous mutation in the Fused in Sarcoma (FUS) gene. ALS6 is a neurodegenerative disorder, which affects the upper and lower motor neurons in the brain and spinal cord, resulting in fatal paralysis. ALS6 is caused by the genetic mutation in the proline/tyrosine-nuclear localization signals of the Fused in sarcoma Protein (FUS). FUS gene also known as TLS (Translocated in liposarcoma), which encodes a protein called RNA-binding protein-Fus (FUS), has a molecular weight of 75?kDa. In this analysis, we applied computational approach to filter the most deleterious and neurodegenerative disease of ALS6-associated mutation on FUS protein. We found H517Q as most deleterious and disease associated using PolyPhen 2.0, I-Mutant 3.0, SIFT, SNPs&GO, PhD-SNP, Pmut, and Mutpred tools. Molecular dynamics simulation (MDS) approach was conducted to investigate conformational changes in the mutant protein structure with respect to its native conformation. MDS results showed the flexibility loss in mutant (H517Q) FUS protein. Due to mutation, FUS protein became more rigid in nature and might alter the structural and functional behavior of protein and play a major role in inducing ALS6. The results obtained from this investigation would help in the field of pharmacogenomics to develop a potent drug target against FUS-associated neurodegenerative diseases.     

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

应用寡核苷酸芯片并行检测CYP1A1和 GSTM1基因多态性

利用寡核苷酸芯片检测方法分析CYP1A1单核苷酸多态性 (SNP)和GSTM1缺失与否 ,实验结果证明了寡核苷酸芯片技术可并行、准确、高效地检测基因的单核苷酸多态性和其他类型的基因多态型 ,可为疾病遗传易感性及单体型的研究提供强有力的研究工具。采用该寡核苷酸芯片 ,检测了 84份正常人的血液DNA样本 ,其中GSTM1基因缺失率达到 4 7 6 % ,接近报道数值。统计分析发现 ,CYP1A1m1 m2的 3种基因型组合TT AG、TT GG和TC GG的发生频率都为 0 ,而根据实验得到的m1和m2各自基因型数据计算 ,它们的发生频率应是11 4 %、2 6 %和 3 1% ,所以推测在所检测的样本中没有T(m1位点 )和G(m2位点 )的连锁组合 ,即m1和m2位点的组合只有 3种单体型 :T A、C A和C G ,其发生频率分别是 6 9 6 %、7 7%和 2 2 6 %。     

Genetic association of single nucleotide polymorphisms in dystrobrevin binding protein 1 gene with schizophrenia in a Malaysian population

Dystrobrevin binding protein 1 (DTNBP1) gene is pivotal in regulating the glutamatergic system. Genetic variants of the DTNBP1 affect cognition and thus may be particularly relevant to schizophrenia. We therefore evaluated the association of six single nucleotide polymorphisms (SNPs) with schizophrenia in a Malaysian population (171 cases; 171 controls). Associations between these six SNPs and schizophrenia were tested in two stages. Association signals with p < 0.05 and minor allele frequency > 0.05 in stage 1 were followed by genotyping the SNPs in a replication phase (stage 2). Genotyping was performed with sequenced specific primer (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). In our sample, we found significant associations between rs2619522 (allele p = 0.002, OR = 1.902, 95%CI = 1.266 – 2.859; genotype p = 0.002) and rs2619528 (allele p = 0.008, OR = 1.606, 95%CI = 1.130 – 2.281; genotype p = 6.18 × 10⁻⁵) and schizophrenia. Given that these two SNPs may be associated with the pathophysiology of schizophrenia, further studies on the other DTNBP1 variants are warranted.     

In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma

In this study, we identified the most deleterious nsSNP in CDKN2A gene through structural and functional properties of its protein (p16INK4A) and investigated its binding affinity with cdk6. Out of 118 SNPs, 14 are nsSNPs in the coding region and 17 SNPs were found in the untranslated region (UTR). FastSNP suggested that 7 SNPs in the 5' UTR might change the protein expression levels. Sixty-four percent of nsSNPs are found to be damaged in PolyPhen server among the 14 nsSNPs investigated. With this effort, we modeled the mutant p16INK4A proteins based on these deleterious nsSNPs, out of which three nsSNPs associated p16INK4A had RMSD values of greater than 3.00 A with native protein. From a comparison of total energy of these three mutant proteins, we identified that the major mutation is from Aspartic acid to Tyrosine at the residue position of 84 of p16INK4A. Further, we compared the binding efficiency of both native and mutant p16INK4A with cdk6. We found that mutant p16INK4A has less binding affinity with cdk6 compared to native type. This is due to ten hydrogen bonds and eight salt bridges which exist between the native type and cdk6, whereas the mutant type makes only nine hydrogen bonds and five salt bridges with cdk6. Based on our investigation, we propose that the SNP with the ID rs11552822 could be the most deleterious nsSNP in CDKN2A gene, causing malignant melanoma, as it was well correlated with experimental studies carried out elsewhere.     

Association of common polymorphisms in the IL2RA gene with type 1 diabetes: evidence of 32,646 individuals from 10 independent studies

Single nucleotide polymorphisms (SNPs) in the interleukin 2 receptor alpha (IL2RA) gene have been suggested to be associated with type 1 diabetes (T1D) susceptibility. However, the results from individual studies are inconsistent. To explore the association of IL2RA polymorphisms with T1D, including rs11594656, rs2104286, rs3118470, rs41295061 and rs706778, a meta‐analysis involving 10 independent studies with 19 outcomes was conducted: five studies with a total of 10,572 cases and 12,956 controls were analysed for rs11594656 with T1D risk, three studies with 7300 cases and 8331 controls for rs2104286, three studies with 3880 cases and 5409 controls for rs3118470, five studies with 11,253 cases and 13,834 controls for rs41295061 and three studies with 1896 cases and 1709 controls for rs706778 respectively. Using minor allelic comparison, the five investigated SNPs were all observed to have a significant association with T1D: For rs11594656, fixed effect model (FEM) odds ratio (OR) 0.87, 95% confidence interval (CI) 0.83, 0.91; rs2104286, FEM OR 0.81, 95% CI 0.77, 0.85; rs3118470, FEM OR 1.23, 95% CI 1.16, 1.31; rs41295061, random effect model (REM) OR 0.67, 95% CI 0.60, 0.76 and rs706778 FEM OR 1.20, 95% CI 1.08, 1.33. Similar results were obtained when all the included studies were calculated by a REM. Our meta‐analysis suggests that all five SNPs in the IL2RA gene are risk factors for T1D risk, and rs11594656, rs2104286 and rs41295061 are the most associated SNPs in the populations investigated. This conclusion warrants confirmation by further studies.     

Cloning and mapping of the porcine cytochrome-p450 2E1 gene and its association with skatole levels in the domestic pig

The porcine cytochrome-p450 2E1 (CYP2E1) gene was isolated by screening a pig BAC library and partially sequenced. This sequence information was used to identify six single nucleotide polymorphisms (SNPs) within the CYP2E1 gene and its promoter. In addition, a microsatellite marker tightly linked to the CYP2E1 gene was subcloned from the BAC. One of these markers was used to map the CYP2E1 gene distal of SWC27 on SSC14, well outside reported quantitative trait loci on SSC14 for skatole, indole and taste test measures of boar taint. However, in a population of commercial pigs scored for backfat skatole levels, there was evidence of association between a SNP in the CYP2E1 promoter and skatole deposition, although there was no significant association between this SNP and skatole levels in the experimental cross.     

Single‐nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu‐opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P_trend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.     

Systems biology approach for mutational and site-specific structural investigation of DNA repair genes for xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a rare genetic skin disorder caused due to the extreme sensitivity for ultraviolet (UV) radiations. On its exposure, DNA acquires damages leading to skin and often neurological abnormalities. The DNA repair implicated in fixing UV-induced damages is NER and mutations in genes involved in NER and TLS form the basis of XP. The analyses of such mutations are vital for understanding XP and involved cancer genetics to facilitate the identification of crucial biomarkers and anticancer therapeutics. We detected the deleterious nsSNPs and examined them at structure-level by altering the structure, estimating secondary structure, solvent accessibility and performing site specific analysis. Crucial phosphorylation sites were also identified for their role in the disorder. These mutational and structural analyses offer valuable insight to the fundamental association of genetic mutations with phenotypic variations in XP and will assist experimental biologists to evaluate the mutations and their impact on genome.     

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.     

Role of BRCA1 and BRCA2 gene mutations in epithelial ovarian cancer in Indian population: a pilot study

Ovarian cancer is a silent killer as most patients have non-specific symptoms and usually present in advanced stage of the disease. It occurs due to certain genetic alterations and mutations namely founder mutations, 187delAG and 5385insC in BRCA1 and 6174delT in BRCA2 which are associated with specific family histories. These highly penetrant susceptibility genes responsible for approximately half of families containing 2 or more ovarian cancer cases account for less than 40% of the familial excess malignancy risk. The remaining risk may be due to single nucleotide polymorphisms (SNPs) which are single base change in a DNA sequence with usual alternatives of two possible nucleotides at a given position. Preliminary study involving 30 women with histologically proven epithelial ovarian cancer was conducted and their detailed genetic analysis was carried out. Regions of founder mutations on BRCA1 and BRCA2 were amplified and sequenced using primers designed based on 200 bp upstream and downstream regions of the mutation sites. Five sequence variants in BRCA1 were identified of which three novel sequence variants were found in 23 patients while in BRCA2, one novel sequence variant was found. The three founder mutations 187delAG, 5385insC in BRCA1 and 6174delT in BRCA2 were not seen in any of the subjects.     

Small-angle X-ray scattering of reduced ribonuclease A: effects of solution conditions and comparisons with a computational model of unfolded proteins

The disulfide-reduced form of bovine ribonuclease A, with the Cys thiols irreversibly blocked, was characterized by small-angle x-ray scattering. To help resolve the conflicting results and interpretations from previous studies of this model unfolded protein, we measured scattering profiles using a range of solution conditions and compared them with the profiles predicted by a computational model for a random-coil polypeptide. Analysis of the simulated and experimental profiles reveals that scattering intensities at intermediate angles, corresponding to interatomic distances in the range of 5-20 Å, are particularly sensitive to changes in solvation and can be used to assess the internal scaling behavior of the polypeptide chain, expressed as a mass fractal dimension, D_m. This region of the scattering curve is also much less sensitive to experimental artifacts than is the very small angle regime (the Guinier region) that has been more typically used to characterize unfolded proteins. The experimental small-angle x-ray scattering profiles closely matched those predicted by the computational model assuming relatively small solvation energies. The scaling behavior of the polypeptide approaches that of a well-solvated polymer under conditions where it has a large net charge and at high urea concentrations. At lower urea concentrations and neutral pH, the behavior of the chain approaches that expected for θ-conditions, where the effects of slightly unfavorable interactions with solvent balance those of excluded volume, leading to scaling behavior comparable to that of an idealized random walk chain. Though detectable, the shift toward more compact conformations at lower urea concentrations does not correspond to a transition to a globule state and is associated with little or no reduction in conformational entropy. This type of collapse, therefore, is unlikely to greatly reduce the conformational search for the native state.