SIFT Analysis Research Articles

Evidence That GRIN2A Mutations in Melanoma Correlate with Decreased Survival

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-d-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 low-passage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought. We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132L would disrupt protein function. When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1–GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P < 0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P = 0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression.

Abstract PD4-2: Whole exome and transcriptome sequencing of 120 primary breast cancer to discover novel therapeutic target

Abstract Introduction: Many somatic mutations, structural alterations, and gene expression changes are causally implicated in oncogenesis and tumor progression, and as a result, affect clinical outcome. Although majority of breast cancer patients have benefits from therapeutics targeting tumor biology, such as estrogen receptor and HER-2, still many patients suffer from disease recurrence and metastasis. More kind of specific target therapies are needed, especially for hormone-resistant tumor and triple-negative breast cancer. Materials and Method: To find novel therapeutic target in breast cancer, here we examine the both whole exome and whole transcriptome of fresh-frozen primary breast cancer tissues from 120 patients whose clinical, pathological, and survival data are available. Patients with Stage IV disease or who received neoadjuvant chemotherapy were excluded. 36 patients had distant metastasis within 5 years from surgery, and 84 patients were NED at least 5 years. RNA and DNA were extracted and qualities were assessed in all samples. Exome and transcriptome sequencing were done using NGS technology (Illumina HiSeq 2000). As a control, exome sequencing was done for 93 normal DNA from matched patients. Single nucleotide variations (SNV) identified in cancer samples on exonic region, nonsynonymous SNV or stop gain/loss, whose quality ≥20, and not found in 93 normal samples were included. SNVs registered in dbSNP135_common or 1000 genome allele frequency &amp;gt;0.001 were excluded.Results and Discussion: We identified 11,684 putative somatic mutations in 7,373 genes. Of them, 6,547 were deleterious or damaging mutation by Provean or SIFT analysis. Mutations were found in potential drug target genes, such as PIK3CA(25), PTEN(3), AKT1(3), ALK(3), ROS1(2), FGFR4(3), FGFR3(2), ERBB2(2), and IDH1(1) etc. In a pathway analysis, mutations in insulin signaling pathway were most dominant. We hypothesized that driver gene and therapeutic target has to have recurrent mutation and gene expression at least more than average expression. We calculated expression “Volume” according to the median normalized FPKM value of individual gene's RNA-seq data. With a cut-off of 3 or more mutations in each gene, 1,116 genes were selected. After the filtering of Volume&amp;lt;0.3, 696 genes were selected. Finally, 55 genes were selected which are druggable or potentially druggable using drug database (DrugBank, TOCRIS, Ingenuity) and Pubmed. DriverNet analysis result was also considered for the selection. All 342 tumor suppressor genes were filtered out. Interestingly, 18 of the 55 were genes involved in metabolism (fatty acid, glucose, amino acids). 12 were kinases and 4 were involved in insulin pathway. Excluding the previously confirmed therapeutic target, PIK3CA, AKT1, and NOTCHs, and considering the patients’ clinical data, our primary candidates for hormone-resistant breast cancer were NQO2, CELSR1, GLUD2, MYH9, PSMD2, NADK, IRS2, MAP3K5, and for triple-negative breast cancer were HSPG2, PHGDH, MYLK, etc. Validation with Sanger sequencing and functional study is on-going. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-2.

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.

Abstract 050: Mutational Spectrum in a Cohort of Chinese Sporadic Dilated Cardiomyopathy by Next-generation Sequencing

Rationale: Dilated cardiomyopathy (DCM) is a leading cause of heart failure and have a genetic basis in 20-30% of cases. Genetic studies in familial DCM have found mutations identified in more than 30 genes. However, the genetics of sporadic DCM is still unknown. Objective: To provide new insights into the pathophysiology of sporadic DCM, a mutational screening on 30 DCM-causing genes in a cohort was performed. Methods and Results: Patients diagnosed with idiopathic DCM were recruited into the study and underwent clinical evaluation. Genomic DNA was isolated from the peripheral blood and then 30 genes ( ACTC, DES, SGCD, MYH7, TNNT2, TPM1, TTN, VCL, MYBPC3, CSRP3, ACTN2, PLN, LDB3, MYH6, ABCC9, TNNC1, TCAP, TNNI3, EYA4, TMPO, PSEN1/2, CRYAB, PDLIM3, MYPN, LAMA4, ILK, ANKRD1, RBM20, LMNA , and SCN5A ) of every sample were detected by next-generation sequencing. All called variants were compared against National Center for Biotechnology Information SNP Database build 137 and the March 2012 release of the 1000 Genomes project. All variants identified in the cohort were compared in ethnic-matched controls consisting of 197 adult Chinese from the 1000 Genomes. The pathogenicity of the variants absent from controls were evaluated by SIFT software. Sixty-six adult Chinese unrelated DCM patients were tested for up to 30 genes causing familial DCM. The mean age was 49.14±15.97 years. Every patient was detected nonsynonymous variants, and the total 87 variants were composed of 39 novel and 48 known. Among the whole 87 variants, 34 satisfied the criteria (1) absent from ethnic-matched controls and (2) altering evolutionary conservation of amino acid verified by SIFT analysis, which were considered pathogenic mutations, identified in 30 patients accounting for 45.45% of the cohort. The 2 most frequent genes involved in mutation positive patients were SCN5A (12.12%) and MYH7 (10.61%). Conclusions: Mutant genes found in familial DCM were also common in sporadic DCM, and the discovery of novel mutations spanned the spectrum of DCM genetics, implicating that genetic etiologies play an important role in pathogenesis of sporadic DCM.

Low‐Density Lipoprotein Receptor Gene Familial Hypercholesterolemia Variant Database: Update and Pathological Assessment

Familial hypercholesterolemia (FH) is caused predominately by variants in the low-density lipoprotein receptor gene (LDLR). We report here an update of the UCL LDLR variant database to include variants reported in the literature and in-house between 2008 and 2010, transfer of the database to LOVDv.2.0 platform (https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR) and pathogenicity analysis. The database now contains over 1288 different variants reported in FH patients: 55% exonic substitutions, 22% exonic small rearrangements (<100 bp), 11% large rearrangements (>100 bp), 2% promoter variants, 10% intronic variants and 1 variant in the 3' untranslated sequence. The distribution and type of newly reported variants closely matches that of the 2008 database, and we have used these variants (n= 223) as a representative sample to assess the utility of standard open access software (PolyPhen, SIFT, refined SIFT, Neural Network Splice Site Prediction Tool, SplicePort and NetGene2) and additional analyses (Single Amino Acid Polymorphism database, analysis of conservation and structure and Mutation Taster) for pathogenicity prediction. In combination, these techniques have enabled us to assign with confidence pathogenic predictions to 8/8 in-frame small rearrangements and 8/9 missense substitutions with previously discordant results from PolyPhen and SIFT analysis. Overall, we conclude that 79% of the reported variants are likely to be disease causing.

A facial sparse descriptor for single image based face recognition

Single image based face recognition under different variations such as occlusion, expression and pose has been recognized as an important task in many real-world applications. The popularly widely used holistic features are easily distorted due to occlusion and some other variations. In order to tackle this problem, the sparse local feature descriptor based recognition methods have become more and more important, and promising performance is obtained. The recently developed SIFT, which detects feature points sparsely and extracts feature locally for object matching between different views and scales, can also benefit single image based face recognition. However, we find in this paper that SIFT should not be directly used for face recognition, because face recognition differs from generic object matching. To this end, we develop a new framework for detecting feature keypoints sparsely, describing feature context and matching feature points between two face images. We call this new proposed framework as Facial Sparse Descriptor (FSD). Experiments are conducted to support our analysis of SIFT, and extensive experiments are also presented to validate the proposed FSD against SIFT and its two variants, two dense local feature descriptor (i.e., LBP and HoG), PCA and Gabor based methods on AR, CMU and FERET databases.

Missense mutation in PFKM associated with muscle-type phosphofructokinase deficiency in the Wachtelhund dog

Hereditary muscle-type phosphofructokinase (PFK) deficiency causing intermittent hemolytic anemia and exertional myopathy due to a single nonsense mutation in PFKM has been previously described in English Springer and American Cocker Spaniels, Whippets, and mixed breed dogs. We report here on a new missense mutation associated with PFK deficiency in Wachtelhunds.Coding regions of the PFKM gene were amplified from genomic DNA and/or cDNA reverse-transcribed from RNA of EDTA blood of PFK-deficient and clinically healthy Wachtelhunds and control dogs. The amplicons were sequenced and compared to the published canine PFKM sequence. A point mutation (c.550C>T, in the coding sequence of PFKM expressed in blood) was found in all 4 affected Wachtelhunds. This missense mutation results in an amino acid substitution of arginine (Arg) to tryptophan (Trp) at position 184 of the protein expressed in blood (p.Arg184Trp). The mutation is located within an alpha-helix, and based on the SIFT analysis, this amino acid substitution is not tolerated. Amplifying the region around this mutation and digesting the PCR fragment with the restriction enzyme MspI, produces fragments that readily differentiate between PFK-deficient, carrier, and normal animals. Furthermore, we document 2 additional upstream PFKM exons expressed in canine testis but not in blood.Despite their similar phenotypic appearance and use for hunting, Wachtelhunds and English Springer Spaniels are not thought to have common ancestors. Thus, it is not surprising that different mutations are responsible for PFK deficiency in these breeds. Knowledge of the molecular basis of PFK deficiency in Wachtelhunds provides an opportunity to screen and control the spread of this deleterious trait.

Mutations in C8orf37, Encoding a Ciliary Protein, are Associated with Autosomal-Recessive Retinal Dystrophies with Early Macular Involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166(∗)]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156-2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156-2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.

Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies Paratuberculosis Infection

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination.

Glutamate receptors: emerging players in melanomagenesis

Glutamate receptors: emerging players in melanomagenesis

A novel mutation in TNNT3 associated with Sheldon–Hall syndrome in a Chinese family with vertical talus

Distal arthrogryposis (DA) is a group of rare, clinically and genetically heterogeneous disorders primarily characterized by congenital contractures of the limb joints. Recently, mutations in genes encoding the fast-twitch skeletal muscle contractile myofibers complex, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosine 2 (TPM2), and embryonic myosin heavy chain 3 (MYH3), and the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1) were confirmed to cause DA1, DA2A, and DA2B. DA2B, or Sheldon–Hall syndrome (SHS; MIM 601680), is intermediate to DA1 and DA2A, or Freeman-Sheldon syndrome (FSS; MIM193700), and shows prominent facial traits. This report describes a Chinese family with SHS over three generations in which all affected individuals showed vertical talus and one demonstrated preauricular tags on the face. Linkage analysis and PCR sequencing revealed a novel substitute mutation at a hot-spot site in TNNT3 (c.187C > T; p.R63C). This mutation was confirmed to cosegregate with the DA phenotype in affected individuals. SIFT and PolyPhen analyses suggest that the mutation is pathogenic. We report this mutation in TNNT3 and speculate that bilateral vertical talus, or severe clubfoot, might be a special characteristic for cases with the TNNT3 R63C mutation.

Two Novel Mutations of the TSH-β Subunit Gene Underlying Congenital Central Hypothyroidism Undetectable in Neonatal TSH Screening

Patients with TSH-beta subunit defects and congenital hypothyroidism are missed by TSH-based neonatal screening. Our objective was to report the molecular consequences of a novel splice-junction mutation and a novel missense mutation in the TSH-beta subunit gene found in two patients with congenital central hypothyroidism and conventional treatment-resistant anemia. Patient 1 had a homozygous G to A nucleotide change at the 5' donor splice site of exon/intron 2. This resulted in a silent change at codon 34 of the mature protein. In vitro splicing assays showed that the mutant minigene dramatically affected pre-mRNA processing, causing exon 2 to be completely skipped. The putative product from a new out-of-frame translational start point in exon 3 is expected to yield a nonsense 25-amino-acid peptide. In patient 2, sequence analysis revealed a compound heterozygosis for the already reported 313delT (C105Vfs114X) mutation and for a second novel mutation in exon 3, substituting G for A at cDNA nucleotide position 323, resulting in a C88Y change. This cysteine residue is conserved among all dimeric pituitary and placental glycoprotein hormone-beta subunits. Data from in silico analysis confirmed that the C88Y mutation would affect subunit conformation. Indeed, two different bioinformatics approaches, PolyPhen and SIFT analysis, predicted C88Y to be a damaging substitution. In isolated TSH deficiency, the exact molecular diagnosis is mandatory for diagnosis of isolated pituitary deficiency, delineation of prognosis, and genetic counseling. Moreover, diagnosis of central hypothyroidism should be considered in the face of severe infant anemia of uncertain etiology.

Novel missense mutations in the ubiquitination‐related gene UBE2A cause a recognizable X‐linked mental retardation syndrome

Recently, a truncating mutation of the UBE2A gene has been observed in a family with X-linked mental retardation (XLMR) (1). The three affected males had similar phenotypes, including seizures, obesity, marked hirsutism and a characteristic facial appearance. Here, we report on two families with a total of seven patients and a clinically very similar syndromic form of XLMR. Linkage analysis was performed in the larger of these families, and screening several positional candidate genes revealed a G23R missense mutation in the UBE2A gene. Subsequent UBE2A screening of a phenotypically similar second family revealed another missense mutation, R11Q, again affecting an evolutionarily conserved amino acid close to the N-terminus of the protein. SIFT and PolyPhen analyses suggest that both mutations are pathogenic, which is supported by their absence in 168 healthy controls. Thus, both missense and truncating mutations can give rise to a specific, syndromic form of XLMR which is identifiable in a clinical setting.

Abstract 2212: A genomic portrait of tumor progression using next-generation sequencing

Abstract Accumulation of genetic and epigenetic alterations is thought to enable cancers to proliferate and survive more effectively than their neighboring cells, or for aggressive tumors to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and chemoresistance is not fully understood. With the advent of next-generation sequencing technologies, we can determine the whole transcriptome or genome sequence of a tumor and thereby detect all the changes that may result in a particular phenotype such as resistance to cytotoxic therapies. To understand the genetic aberrations underlying the chemoresistance in neuroblastoma, we attempted to catalogue all genetic aberrations in two tumor samples before and after cytotoxic therapies using RNA-seq (including whole transcriptome and miRNA) and genomic DNA sequencing from the same neuroblastoma patient who was refractory to standard chemotherapies. First, we performed whole genome sequencing on patient's constitutional DNA to establish a genetic background. Multiple mate-paired libraries were constructed and subjected to next-generation sequencing. We acquired 53 Gb high-quality mappable data, equivalent to 16X coverage of this patient's genome. Next, whole transcriptome sequencing of the two tumors yielded total 345 million 50bp mappable reads representing 14,128 and 14,484 genes (20875 and 21330 transcripts) respectively. The average coverage for each transcript was &amp;gt;14, while 5297 genes for one sample and 6912 genes for the other had an average coverage of &amp;gt;10. With such deep sequencing, single nucleotide variant (SNV) analysis on the transcriptome sequencing data identified 9103 and 10556 exonic SNVs of total 154871 and 191033 SNVs respectively in two samples. Among the exonic SNVs, 3965 and 4470 SNVs were in the coding regions and about half of them were nonsynonymous. Using SIFT analysis, we predicted 353 and 443 SNVs to be damaging before and after chemotherapies. Of them, 165 SNVs were shared among two samples. Therefore, these shared SNVs and newly acquired SNVs after chemotherapies may contain the genetic alterations that are resistant to the standard neuroblastoma therapies in this patient. In addition, 188 SNVs were present in the original tumor but not in the refractory sample indicating genomic instability of tumors. In parallel, we performed microRNA sequencing on the two samples. While most of 987 mature microRNAs were detected at similar levels after normalized against total reads, 64 microRNAs were significantly differentially expressed before and after chemotherapies using stringent selection criteria (total read counts in two samples&amp;gt;500, &amp;gt;5 fold change, P&amp;lt;0.01, and FDR&amp;lt;0.01). These differentially microRNAs and accumulation of deleterious mutations during therapies are currently being investigated for pathway disruption that may lead to chemo-refractory disease and allow development of targeted therapies to overcome drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2212.

Diversity and evolution of 11 innate immune genes in Bos taurus taurus and Bos taurus indicus cattle

The Toll-like receptor (TLR) and peptidoglycan recognition protein 1 (PGLYRP1) genes play key roles in the innate immune systems of mammals. While the TLRs recognize a variety of invading pathogens and induce innate immune responses, PGLYRP1 is directly microbicidal. We used custom allele-specific assays to genotype and validate 220 diallelic variants, including 54 nonsynonymous SNPs in 11 bovine innate immune genes (TLR1-TLR10, PGLYRP1) for 37 cattle breeds. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and we were unable to differentiate between the specialized B. t. taurus beef and dairy breeds, despite an average polymorphism density of one locus per 219 bp. Ninety-nine tagSNPs and one tag insertion-deletion polymorphism were sufficient to predict 100% of the variation at all 11 innate immune loci in both subspecies and their hybrids, whereas 58 tagSNPs captured 100% of the variation at 172 loci in B. t. taurus. PolyPhen and SIFT analyses of nonsynonymous SNPs encoding amino acid replacements indicated that the majority of these substitutions were benign, but up to 31% were expected to potentially impact protein function. Several diversity-based tests provided support for strong purifying selection acting on TLR10 in B. t. taurus cattle. These results will broadly impact efforts related to bovine translational genomics.

Characterization of the transcriptional regulation of the human MT1-MMP gene and association of risk reduction for focal-segmental glomerulosclerosis with two functional promoter SNPs

The matrix metalloproteinase MT1-MMP (MMP-14) is an important player in wound healing, bone development, angiogenesis, inflammation and tumour invasion. MT1-MMP also plays an important role in the development and resolution of experimental kidney diseases. The role of MT1-MMP was investigated for distinction between minimal-change glomerulonephritis (MCGN) and focal-segmental glomerulosclerosis (FSGS) that can sometimes be difficult due to sampling error in renal biopsy. We defined the transcriptional regulation of the human MT1-MMP and the influence of single nucleotide polymorphisms (SNPs) within its promoter region in renal mesangial cells with reporter gene constructs and gel sift analysis. Genomic DNA from healthy blood donors (n = 500) and from kidney biopsies with defined renal diseases (MCGN: n = 189, FSGS: n = 311) was screened for MT1-MMP promoter SNPs. Transcription of MT1-MMP is regulated by two enhancers, an Sp1 binding site and a regulatory region 1 (RR1). RR1 contains an Ets site binding the transcription factors Elf-1 and E1AF but not NFAT. The MT1-MMP promoter contains two SNPs (-378 T/C and -364 G/T) in close vicinity to the RR1. Occurrence of the SNP variant -378 C leads to strong inhibition of nuclear protein binding to the RR1 reducing its enhancer function. Appearance of either variant -378 C or variant -364 T in at least one copy of the MT1-MMP promoter was associated with a significant risk reduction for the development of FSGS (P < 0.048). Genetic testing for MT1-MMP promoter SNPs could put renal biopsy results into new perspective. An independent study will be required to verify these findings and their possible diagnostic value for differentiation between certain renal diseases.

Pattern of sequence variation across 213 environmental response genes.

To promote the clinical and epidemiological studies that improve our understanding of human genetic susceptibility to environmental exposure, the Environmental Genome Project (EGP) has scanned 213 environmental response genes involved in DNA repair, cell cycle regulation, apoptosis, and metabolism for single nucleotide polymorphisms (SNPs). Many of these genes have been implicated by loss-of-function mutations associated with severe diseases attributable to decreased protection of genomic integrity. Therefore, the hypothesis for these studies is that individuals with functionally significant polymorphisms within these genes may be particularly susceptible to genotoxic environmental agents. On average, 20.4 kb of baseline genomic sequence or 86% of each gene, including a substantial amount of introns, all exons, and 1.3 kb upstream and downstream, were scanned for variations in the 90 samples of the Polymorphism Discovery Resource panel. The average nucleotide diversity across the 4.2 MB of these 213 genes is 6.7 x 10(-4), or one SNP every 1500 bp, when two random chromosomes are compared. The average candidate environmental response gene contains 26 PHASE inferred haplotypes, 34 common SNPs, 6.2 coding SNPs (cSNPs), and 2.5 nonsynonymous cSNPs. SIFT and Polyphen analysis of 541 nonsynonymous cSNPs identified 57 potentially deleterious SNPs. An additional eight polymorphisms predict altered protein translation. Because these genes represent 1% of all known human genes, extrapolation from these data predicts the total genomic set of cSNPs, nonsynonymous cSNPs, and potentially deleterious nonsynonymous cSNPs. The implications for the use of these data in direct and indirect association studies of environmentally induced diseases are discussed.

The validation of in vitro methods for skin irritation

Several alternative in vitro methods for identifying skin irritants have been developed in the last 10 years, the most promising of which use either reconstituted human skin models or animal (e.g. pig or mouse) skin organ cultures. In 1998, the European Centre for the Validation of Alternative Methods (ECVAM) commissioned a pre-validation study of five methods. Two reconstituted human skin methods (Epiderm and EPISKIN) and one animal skin model (the mouse skin integrity function test, SIFT) performed well in Phases I and II of the study (intralaboratory reproducibility and protocol transfer) and proceeded to Phase III which assessed reproducibility and predictive ability in three independent laboratories using a set of 20 coded test chemicals (10 irritants and 10 non irritants). Intralaboratory reproducibility was again acceptable for all three methods but inter-laboratory reproducibility was acceptable only for EPISKIN. The predictive ability of all three methods was also inadequate. Following refinements to the protocols of Epiderm and EPISKIN and changes to the statistical analysis of SIFT, the predictive ability was improved. In 2003, ECVAM concluded that they could proceed to a full validation study. This will be conducted in two phases and is scheduled for completion early in 2005.