Igor Vorechovsky

Mutations of the Human Homolog of Drosophila patched in the Nevoid Basal Cell Carcinoma Syndrome

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

A Mammalian patched Homolog Is Expressed in Target Tissues of sonic hedgehog and Maps to a Region Associated with Developmental Abnormalities

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

Low frequency of alterations of the α (PPP2R1A) and β (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms

The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the β isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both α (PPP2R1A) and β isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.

Fine-Scale Mapping at IGAD1 and Genome-Wide Genetic Linkage Analysis Implicate HLA-DQ/DR as a Major Susceptibility Locus in Selective IgA Deficiency and Common Variable Immunodeficiency

Selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID) are the most common primary immunodeficiencies in humans. A high degree of familial clustering, marked differences in the population prevalence among ethnic groups, association of IgAD and CVID in families, and a predominant inheritance pattern in multiple-case pedigrees have suggested a strong, shared genetic predisposition. Previous genetic linkage, case-control, and family-based association studies mapped an IgAD/CVID susceptibility locus, designated IGAD1, to the MHC, but its precise location within the MHC has been controversial. We have analyzed a sample of 101 multiple- and 110 single-case families using 36 markers at the IGAD1 candidate region and mapped homozygous stretches across the MHC shared by affected family members. Haplotype analysis, linkage disequilibrium, and homozygosity mapping indicated that HLA-DQ/DR is the major IGAD1 locus, strongly suggesting the autoimmune pathogenesis of IgAD/CVID. This is supported by the highest excess of allelic sharing at 6p in the genome-wide linkage analysis of 101 IgAD/CVID families using 383 marker loci, by previously reported restrictions of the T cell repertoires in CVID, the presence of autoantibodies, impaired T cell activation, and a dysregulation of a number of genes in the targeted immune system. IgAD/CVID may thus provide a useful model for the study of pathogenesis and novel therapeutic strategies in autoimmune diseases.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

Transposable elements in disease-associated cryptic exons

Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon–intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.

Does 77C-->G in PTPRC modify autoimmune disorders linked to the major histocompatibility locus?

A 77G allele of the gene encoding CD45, also known as the protein tyrosine phosphatase receptor-type C gene (PTPRC), has been associated with multiple sclerosis (MS). Here we determine allele frequencies in large numbers of MS patients, primary immunodeficiencies linked to the major histocompatibility complex (MHC) locus and over 1,000 controls to assess whether aberrant splicing of PTPRC caused by the 77C→G polymorphism results in increased susceptibility to these diseases. Our results show no difference in the frequency of the 77G allele in patients and controls and thus do not support a causative role for the polymorphism in the development of disorders with a strong autoimmune component in etiology.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38–50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6

Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among disease‐causing exonic substitutions is unknown. Here, we have tested missense and silent mutations deposited in the BRCA1 mutation databases of unclassified variants for their effects on exon inclusion. Analysis of 21 BRCA1 variants using minigene assays revealed a single exon‐skipping mutation c.231G>T. Comprehensive mutagenesis of an adjacent 12‐nt segment showed that this silent mutation resulted in a higher level of exon skipping than the 35 other single‐nucleotide substitutions. Exon inclusion levels of mutant constructs correlated significantly with predicted splicing enhancers/silencers, prompting the development of two online utilities freely available at http://www.dbass.org.uk. EX‐SKIP quickly estimates which allele is more susceptible to exon skipping, whereas HOT‐SKIP examines all possible mutations at each exon position and identifies candidate exon‐skipping positions/substitutions. We demonstrate that the distribution of exon‐skipping and disease‐associated substitutions previously identified in coding regions was biased toward top‐ranking HOT‐SKIP mutations. Finally, we show that proteins 9G8, SC35, SF2/ASF, Tra2, and hnRNP A1 were associated with significant alterations of BRCA1 exon 6 inclusion in the mRNA. Together, these results facilitate prediction of exonic substitutions that reduce exon inclusion in mature transcripts. Hum Mutat 32:1–9, 2011.

Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

Mutations that affect splicing of precursor messenger RNAs play a major role in the development of hereditary diseases. Most splicing mutations have been found to eliminate GT or AG dinucleotides that define the 5′ and 3′ ends of introns, leading to exon skipping or cryptic splice-site activation. Although accurate description of the mis-spliced transcripts is critical for predicting phenotypic consequences of these alterations, their exact nature in affected individuals cannot often be determined experimentally. Using a comprehensive collection of exons that sustained cryptic splice-site activation or were skipped as a result of splice-site mutations, we have developed a multivariate logistic discrimination procedure that distinguishes the two aberrant splicing outcomes from DNA sequences. The new algorithm was validated using an independent sample of exons and implemented as a free online utility termed CRYP-SKIP (http://www.dbass.org.uk/cryp-skip/). The web application takes up one or more mutated alleles, each consisting of one exon and flanking intronic sequences, and provides a list of important predictor variables and their values, the overall probability of activating cryptic splice vs exon skipping, and the location and intrinsic strength of predicted cryptic splice sites in the input sequence. These results will facilitate phenotypic prediction of splicing mutations and provide further insights into splicing enhancer and silencer elements and their relative importance for splice-site selection in vivo.