Unique Deletion Research Articles

Identification of four novel developmentally regulated gamma hemoglobin mRNA isoforms

In the course of developing assays for the molecular prediction of biological age, we serendipitously discovered four novel isoforms of gamma hemoglobin mRNA, designated HBG1n1, HBG1n2, HBG2n2, and HBG2n3, collectively termed HBGn isoforms. Here we report the molecular characterization and tissue expression of these isoforms. RNA obtained from human peripheral blood and various fetal and adult tissues was amplified with duplex reverse transcription polymerase chain reaction (RT-PCR) assays to determine the expression profiles of the HBGn isoforms. To determine if their molecular origin was either a genomic recombination or an undefined RNA rearrangement event, DNA and RNA samples were pretreated with RNaseI and/or DNaseI and then analyzed with the duplex RT-PCR assays. Alignment analysis indicated that the HBG1n(1/2) and HBG2n(2/3) isoforms were identical to the expected parental HBG1 or HBG2 sequences except for unique deletions that spanned the 3' end of exon two, the entire second intron, and the 5' end of exon three. RT-PCR duplex reactions revealed that the isoforms exhibit restricted expression to fetal hematopoietic tissue and newborn peripheral blood and appear to be temporally regulated during development. Finally, nucleic acid digestion experiments illustrate that the isoforms appear to be created by an RNA rearrangement event between penta- or octanucleotide direct repeats in adjacent exons. The precise genesis and function of these novel isoforms is unknown at present. Interestingly, each of the four isoforms identified maintain an open reading frame as well as 5' and 3' regulatory regions invoking the possibility that they encode a family of related polypeptides.

Proximal interstitial 1p36 deletion syndrome: The most proximal 3.5-Mb microdeletion identified on a dysmorphic and mentally retarded patient with inv(3)(p14.1q26.2)

From the investigation by microarray-based comparative genomic hybridization (aCGH), a new syndrome with “atypical” proximal interstitial deletion of 1p36.23-36.11 has been suggested. Here, we report on an 8.5-year-old girl with psychomotor developmental delay and a dysmorphic appearance. Although her G-banded chromosomal analysis showed inv(3)(p14.1q26.2), detailed FISH analyses denied pathogenic deletions around the breakpoints of chromosome 3. Accordingly, aCGH analysis was performed to identify a genomic aberration related to her phenotype, and a 3.5-Mb interstitial deletion of 1p36.13-36.12 was revealed. This deletion was the most proximal interstitial deletion of 1p36. Compared to the previously reported patients, abnormally shaped teeth, delayed tooth eruption, and leg malformation are unique phenotypes only to this patient, which might be due to the centromeric unique deletion region with 0.8-Mb.

Frequent Detection of Merkel Cell Polyomavirus in Human Merkel Cell Carcinomas and Identification of a Unique Deletion in the VP1 Gene

Merkel cell carcinoma (MCC) is a rare but very aggressive human malignancy of the elderly or immunosuppressed patients. Recently, the clonal integration of a new human polyoma virus, which was termed Merkel cell polyomavirus (MCPyV), has been reported in 8 of 10 MCC patients. In the present study, we studied the formalin-fixed and paraffin-embedded tissue specimens of 39 MCC for the presence of MCPyV by PCR. We applied four different primer sets directed against the large T antigen and the VP1 gene of MCPyV. We were able to detect MCPyV in 77% (n = 30) of MCC as confirmed by sequence analyses of the PCR products. Sequence analyses showed only minor nucleotide changes compared with the previously published MCC sequences. In addition, one patient revealed the amplification of two PCR products using PCR primers directed against the VP1 gene. Sequence analyses confirmed the presence of the expected 351-bp PCR product and in addition a second PCR product of 261 bp containing a unique 90-bp deletion in the VP1 gene, which will lead to a predicted loss of 28 amino acids. The unique 90-bp deletion within the VP1 gene possibly is a result of incomplete viral integration of MCPyV in the MCC. The presence of MCPyV in the majority of MCC tissue specimens in our study strongly underlines a possible role for MCPyV as an etiologic agent in the carcinogenesis of MCC.

Distribution and Evolution of Pseudogenes, Gene Losses, and a Gene Rearrangement in the Plastid Genome of the Nonphotosynthetic Liverwort, Aneura mirabilis (Metzgeriales, Jungermanniopsida)

The plastid genome sequence of the parasitic liverwort Aneura mirabilis revealed the loss of five chlororespiration (ndh) genes. Additionally, six ndh genes, subunits of photosystem I, photosystem II, and the cytochrome b6f complex were inferred to be pseudogenes. Pseudogenes of cysA, cyst, ccsA, and ycf3, an inversion of psbE and petL, were also detected. The designation of pseudogenes was made using comparisons with the distantly related liverwort Marchantia polymorpha. We sampled several populations of A. mirabilis and its photosynthetic sister groups to correlate functional gene losses with the evolution of a achlorophylly. The gene losses, pseudogenes, or the psbE-petL inversion were never detected in a photosynthetic Aneura but were detected in every population of A. mirabilis. One population of A. mirabilis revealed a unique deletion of 541 bp in the psbE-petL region; another is characterized by a unique deletion of 471 bp in the trnV(UAC)-ndhC region. The ratio of synonymous-to-nonsynonymous substitution rates (omega) was estimated for eight pseudogenes and six ORFs to detect relaxed purifying selection. A significant increase in omega for the nonphotosynthetic liverwort was detected in six pseudogenes. Relaxation purifying selection, determined by a significant increase in omega, was detected for three intact ORFs: psbA, psbM, and rbcL.

Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors

BackgroundMicroarray Comparative Genomic Hybridization (array CGH) provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes.ResultsWe evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from .ConclusionWe conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.

Properties and cDNA cloning of antihemorrhagic factors in sera of Chinese and Japanese mamushi ( Gloydius blomhoffi)

An antihemorrhagic protein has been isolated from the serum of Chinese mamushi ( Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein—designated Chinese mamushi serum factor (cMSF)—suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu ( Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi ( G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.

Genetic polymorphism in collagen type 1 alpha 2 intron 33 can be used for the initial screening of East Asians quickly and easily by a heteroduplex analysis

To save time and simplify the matching genetic information of bereaved family with the collected samples for identification of individuals in a mass-scale disaster and terrorism using molecular biological methods, we propose a method to initially screen samples according to different populations. We found a unique deletion (26822–3) in an allele that 62.0% of East Asian individuals had in intron 33 of type I collagen alpha 2 (COL1A2), while most Europeans and Africans did not have it. By making a heteroduplex complex, a visual discrimination was possible using 10% PAGE. A PCR and heteroduplex analysis could identify genetic polymorphism of COL1A2 intron 33 both clearly and accurately, even from decomposed samples or very small amounts of samples within a very short time period (approximately within 150 min). As a result, genetic polymorphism of COL1A2 intron 33 is thus considered to be useful to quickly and easily screen East Asians from vast number of samples using a heteroduplex analysis. Consequently, we consider that an initial screening method for East Asians which use a heteroduplex analysis of genetic polymorphism of COL1A2 intron 33 can smoothly and effectively perform various identifications among East Asians when supplies are logistically poor after a mass-scale disaster or terrorism.

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.

Two Distinct Isolates of Tomato yellow leaf curl virus Threaten Tomato Production in Arizona and Sonora, Mexico.

Severe yellow leaf curl and plant stunting symptoms were observed in tomato plants from two home gardens in central Arizona (Phoenix area) and a tomato field in Sonora, Mexico during the fall of 2006. Disease symptoms were reminiscent of those reported in Florida during 1994 (4) and more recently in tomato fields in the Pacific Coast state of Sinaloa, Mexico found to be infected with the exotic Tomato yellow leaf curl virus (TYLCV) (2). Total DNA was extracted from two symptomatic tomato plants from Arizona and Sonora and used as a template in PCR. PCR products of the core region of the begomovirus coat protein gene (Cp) were cloned (n = 3) and the DNA sequence was determined. BLAST analysis of the 579 bases with sequences available in the NCBI GenBank database indicated the closest match was to an isolate of the monopartite begomovirus TYLCV from Israel, which was known to have been introduced into the Caribbean region, including Puerto Rico, the southeastern United States, and Mexico from 1990 to 1996 (1,4). The full-length TYLCV genome (approximately 2,800 bases) was amplified for a field isolate from each location by rolling circle amplification (RCA) using TempliPhi (Amersham Biosciences, Piscataway, NJ). RCA products were cloned into the plasmid vector pGEM7 (Promega, Madison, WI) that had been previously digested with SacI endonuclease. The complete TYLCV genome sequence was determined for six clones from each RCA product. Nucleotide analysis indicated that the complete TYLCV genome sequences from Sonora and Arizona, respectively, shared 97.6 and 97.7% nt identity. The comparative sequence analysis indicated that TYLCV-Sonora (TYLCV-Son) (GenBank Accession No. EF210555) was 99.1% nt identical to TYLCV reported recently from Culiacan, Mexico (GenBank Accession No. DQ631892). In contrast, TYLCV-AZ (GenBank Accession No. EF210554) shared 99.3% identity with an isolate from Texas, TYLCV-TX (GenBank Accession No. EF110890) (3). Interestingly, the TX and AZ TYLCV isolates contained a unique 29-nt deletion in the intergenic region (IR) between the TATA-box and the nonanucleotide, initiating at nt coordinate 2696. Except for the deletion in the IR region of the AZ and TX isolates, these viruses shared 97.6 to 99.1% nt identity to other TYLCV isolates reported in the Western Hemisphere. The genome sequence for TYLCV-Son shares high nt identity with TYLCV isolates identified in the Yucatan Peninsula and Pacific Coast of Mexico (2), the Caribbean region, and the southeastern United States, suggesting that a single TYLCV species was introduced and has spread throughout North America and the Caribbean (4). The absence of other TYLCV isolates in the Western Hemisphere with the novel 29-nt deletion noted for the TX and AZ isolates suggests that the latter two isolates originated from the same U.S. source. In Mexico, TYLCV was first introduced in the east coast and Yucatan region approximately in 1996. From there, this isolate has spread to the western part of the country (Sinaloa and Sonora) from 2004 to 2006 (2). Similarly, in the United States, TYLCV was introduced and spread in the eastern U.S. states beginning in 1994 (4), where it had been confined until it was discovered in Texas (3) and now Arizona during 2006.

Whole‐genome array‐CGH identifies novel contiguous gene deletions and duplications associated with developmental delay, mental retardation, and dysmorphic features

Cytogenetic imbalances are the most frequently identified cause of developmental delay or mental retardation, which affect 1-3% of children and are often seen in conjunction with growth retardation, dysmorphic features, and various congenital anomalies. A substantial number of patients with developmental delay or mental retardation are predicted to have cytogenetic imbalances, but conventional methods for identifying these imbalances yield positive results in only a small fraction of these patients. We used microarray-based comparative genomic hybridization (aCGH) to study a panel of 20 patients predicted to have chromosomal aberrations based on clinical presentation of developmental delay or mental retardation, growth delay, dysmorphic features, and/or congenital anomalies. Previous G-banded karyotypes and fluorescence in situ hybridization results were normal for all of these patients. Using both oligonucleotide-based and bacterial artificial chromosome (BAC)-based arrays on the same panel of patients, we identified 10 unique deletions and duplications ranging in size from 280 kb to 8.3 Mb. The whole-genome oligonucleotide arrays identified nearly twice as many imbalances as did the lower-resolution whole-genome BAC arrays. This has implications for using aCGH in a clinical setting. Analysis of parental DNA samples indicated that most of the imbalances had occurred de novo. Moreover, seven of the 10 imbalances represented novel disorders, adding to an increasing number of conditions caused by large-scale deletions or duplications. These results underscore the strength of high-resolution genomic arrays in diagnosing cases of unknown genetic etiology and suggest that contiguous genomic alterations are the underlying pathogenic cause of a significant number of cases of developmental delay.

Phylogenetic reconstructions in the genus Capra (Bovidae, Artiodactyla) based on the mitochondrial DNA analysis

Mitochondrial genome fragments were examined in all species of the genus Capra (Bovidae, Artiodactyla). Phylogenetic analysis was carried out using 59 cytochrome b gene sequences (392 bp), and 22 sequences of the mtDNA variable fragment (402 bp). In the control region, two unique deletions were revealed. One of the deletions was found only in Capra cilindricornis (17 bp), while another one grouped C. caucasica with C. aegagrus (1 bp). The group of Caucasian wild goats splits into two clades, and furthermore, the sequences of C. caucasica demonstrate remarkable similarity to the sequences of C. aegagrus, while C. cylindricus seems to have evolved independently for a long period of time. It was demonstrated that C. pyrenaica and C. ibex were extremely close to one another. Capra sibirica formed an outer group relative to the other species, and according to our data, was the most ancient species of the genus. On the contrary, genetic distance separating C. falconeri (the most independent species of the genus related to its morphology) from the other species is small.

HIV and liver disease forum: Conference proceedings

Liver disease has emerged as one of the major causes of morbidity and mortality among patients infected with the human immunodeficiency virus (HIV), particularly in regions where highly active antiretroviral therapy (ART) is widely available. This dramatic change in disease epidemiology is attributable to a complex interaction between etiologic factors that appear to increase the rate of hepatic fibrosis and accelerate progression to end-stage liver disease. Key factors include ART related hepatoxicity, frequent coinfection with hepatitis B and C, and possibly the direct interaction of HIV virus or soluble protein viral products that interact with hepatocytes and other liver resident cell types. Additionally, there is some evidence that gut permeability is altered during active HIV replication, which affects the complex mix of toxins and growth factors present in the portal circulation. It is critical that hepatologists maintain a strong knowledge base to provide the best possible guidance to HIV-infected patients and their healthcare providers. To this end, the second international forum on HIV and Liver Disease was convened in Jackson Hole, WY in September 2008. The first forum, held two years earlier was previously summarized in Hepatology, and has been widely cited by experts in the field.1 However, the fast moving nature of this critical health issue led to development of a second meeting, supported by grants from three institutes of the National Institutes of Health (NIAID, NIDDK, NIAAA) and by unrestricted grants provided by the pharmaceutical industry. As before, the meeting sought to bring together basic and clinical researchers representing multiple disciplines including hepatology, infectious diseases, epidemiology, virology, and drug development as well as governmental experts in health policy, research and research funding. This document provides a summary of key presentations and highlights the current state of knowledge and future directions this field will take.

Possible Role for Mutations in the Transactivation Domain of C-MYC in the Clinical Transformation of Follicular Lymphomas.

Introduction: Follicular lymphoma (FL) is the second most prevalent non-Hodgkins lymphoma (NHL) in the United States. Although the clinical course is usually indolent, about 50% of low-grade FL transform to an aggressive lymphoma within 15 years of diagnosis. Somatic hypermutation (SHM) is a normal process in B cells which targets the immunoglobulin locus and introduces point mutations and small insertions/deletions. In diffuse large B cell lymphoma, the SHM misfires and aberrantly targets multiple loci, including some proto-oncogenes (PIM-1, PAX-5, RhoH/TTF and C-MYC).Hypothesis: We hypothesized that acquired mutations in the same proto-oncogenes could be responsible for the clinical transformation of follicular lymphoma.Materials and methods: The clinical database of the Department of Pathology, Washington University School of Medicine, St. Louis was searched to find patients with serial biopsies and the diagnosis of either stable (sFL) or transformed follicular lymphoma (tFL). Thirty-five (35) formalin-fixed paraffin-embedded (FFPE) tissue samples from 17 patients were identified. Eleven of seventeen patients had transformed to large cell lymphoma and six had stable low-grade follicular lymphoma. To exclude the possibility of a second unrelated lymphoma the common clonal origin of the original and follow-up lymphoma was confirmed by comparing immunoglobulin heavy and/or light chain variable gene sequences. Previously identified mutational hot spots in four genes, BCL-6, PAX-5, RhoH/TTF and C-MYC, were amplified and sequenced directly.Results: Fifty-five mutations were found in the four genes sequenced; all patients had at least one mutation. The number of mutations was similar in both the low grade and the transformed specimens overall, and most paired specimens had identical mutations or a single mutation change over time. Of the mutations identified, only mutations in Myc exon 2 correlated with the transformation. Acquired missense mutations were detected in the trans-activation domain of Myc in two of the eleven transformed FL specimens but none of the 24 low-grade follicular lymphoma specimens. The first case had two mutations: Pro57 to Ser57 and Glu39 to Asp39. The second case had a single Glu39 to Asp39 change. One of the mutations (Pro57 to Ser57) is close to a functionally critical phosphorylation site, Thr58, and has been reported in both Diffuse large B cell and Burkitt's lymphomas. The other mutation (Glu39 to Asp39) has also been reported in both Burkitts and DLBCL cases, and is not reported as a normal variant.Conclusion: There is not an accumulation in the overall number of SHM with transformation as the frequency of SHM is similar in low-grade FL and transformed FL. However, mutations in the trans-activation domain in c-Myc, caused by an aberrant SHM, may play a role in the transformation of low-grade FL to a diffuse, large cell lymphoma in a subset of patients.Somatic Hypermutation Analysis of 17 serial Follicular Lymphoma cases (35 specimens)Gene Name% Specimens with SHMMutation frequency per 100 bpNumber of unique single base pair substitutionsNumber of unique deletions or insertionsNumber of substitutions targeting G+C vs A+TBCL-663%0.1425316 vs 9PAX-543%0.06727 vs 0Rho/TTF17%0.03821 vs 7C-MYC31%0.02616 vs 0All genes83%NA47831 vs 17NA = Not applicable

Patient with novel interstitial deletion of chromosome 3q13.1q13.3 and agenesis of the corpus callosum

Interstitial deletions of the proximal long arm of chromosome 3 are rare. Only eight previously reported patients have deletions involving the proximal segment of 3q. Of these patients, three had agenesis of the corpus callosum and one had holoprosencephaly. We report here a patient with a small unique interstitial deletion of the long arm of chromosome 3 spanning 3q13.1q13.3. This patient has agenesis of the corpus callosum, global developmental delay, and distinctive facial features of a small nose, anteverted nares, and broad nasal root. Our patient provides further evidence that a gene involved in corpus callosum development or neuronal migration may reside in this region.

A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

A granule cell neuron-associated JC virus variant has a unique deletion in the VP1 gene

The human polyomavirus JC (JCV) typically infects glial cells and is the aetiological agent of progressive multifocal leukoencephalopathy (PML), which occurs in immunosuppressed individuals. The full-length sequence of a granule cell neuron-tropic JCV variant, JCV(GCN1), associated with lytic infection of granule cell neurons and cerebellar atrophy in a human immunodeficiency virus-infected patient with PML was determined and compared with the sequence of the JCV isolate from the classic PML lesions present in the hemispheric white matter of the same individual (JCV(HWM)). A unique deletion was found in the C terminus of the VP1 gene of JCV(GCN1), which encodes the major capsid protein, resulting in a frame shift and a total change of the C-terminal amino acid sequence of this protein. This deletion was not present in JCV(HWM), suggesting that this mutation may be instrumental in facilitating entry or replication of JCV into granule cell neurons.

SOD1: A Candidate Gene for Keratoconus

To screen superoxide dismutase 1 (SOD1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC). Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the SOD1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced. A heterozygous genomic 7-base deletion in intron 2 of the SOD1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in 312 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn SOD (SOD1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon 3 (LE2E3). A unique genomic deletion within intron 2 close to the 5' splice junction of the SOD1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E3) that others have shown to code for proteins lacking the active site of the SOD1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.

Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing

Wilson disease is an autosomal recessive inherited disorder of copper metabolism. The Wilson disease gene codes for a copper transporting P-type ATPase (ATP7B). Molecular genetic analysis reveals at least 300 distinct mutations. While most reported mutations occur in single families, a few are more common. The most common mutation in patients from Central, Eastern, and Northern Europe is the point mutation H1069Q (exon 14). About 50-80% of Wilson disease (WD) patients from these countries carry at least one allele with this mutation with an allele frequency ranging between 30 and 70%. Other common mutations in Central and Eastern Europe are located on exon 8 (2299insC, G710S), exon 15 (3400delC) and exon 13 (R969Q). The allele frequency of these mutations is lower than 10%. In Mediterranean countries there is a wide range of mutations, the frequency of each of them varies considerably from country to country. In Sardinia, a unique deletion in the 5' UTR (-441/-427 del) is very frequent. In mainland Spain the missense mutation M645R in exon 6 is particularly common. Data from non-European countries are scarce. Most data from Asia are from Far Eastern areas (China, South Korea and Japan) where the R778L missense mutation in exon 8 is found with an allele frequency of 14-49%. In summary, given the constant improvement of analytic tools genetic testing will become an integral part for the diagnosis of WD. Knowledge of the differences in the worldwide distribution of particular mutations will help to design shortcuts for genetic diagnosis of WD.

A phylogenetic analysis of the major groups of catfishes (Teleostei: Siluriformes) using rag1 and rag2 nuclear gene sequences

Higher-level relationships among catfishes were investigated by parsimony, maximum likelihood and Bayesian analyses of two nuclear genes across 110 catfish species representing 36 of 37 families and Conorhynchos (family incertae sedis). Analysis of 3660 aligned base pairs from the rag1 and rag2 genes confirms monophyly of Siluriformes, of most siluriform families and of a number of multifamily groups, some recognized, some novel. South American Loricarioidei are recovered as the sistergroup to other catfishes which are divided into Diplomystidae and Siluroidei. This result contrasts with the prevailing hypothesis that Diplomystidae is the sister to all other catfishes. Monophyly of Siluroidei is supported by rag data including a unique three-codon deletion from rag1. Deep within Siluroidei are 12 large, strongly supported groups with poorly resolved interrelationships. Five are single families: Cetopsidae, Plotosidae, Chacidae, Siluridae and Pangasiidae. Four others are monophyletic taxa ranked here as superfamilies: Clarioidea (Clariidae, Heteropneustidae), Arioidea (Ariidae, Anchariidae), Pimelodoidea (Pimelodidae, Pseudopimelodidae, Heptapteridae, Conorhynchos), Ictaluroidea (Ictaluridae, Cranoglanididae). South American Doradoidea (Doradidae, Auchenipteridae) and Aspredinidae are a sistergroup pair. Sisoroidea (without Aspredinidae), Ailia+Laides, Horabagridae, and Bagridae (without Rita) form a large, predominantly Asian clade, "Big Asia." Mochokidae, Malapteruridae, Amphiliidae, Claroteidae, and African schilbids are united as a species-rich African clade, "Big Africa." The three large continental clades, "Big Asia," "Big Africa" and Neotropical Loricarioidei suggest a prevalence of intracontinental diversification of catfishes. South America is the home of the Gymnotiformes, putative sistergroup of catfishes, plus two of the deepest siluriform clades, Loricarioidei and Diplomystidae, thus suggesting an ancient siluriform presence if not origin there. The rag phylogeny does not identify any African-South American catfish clade. The well-known African-Asian relationships within families Clariidae and Bagridae are confirmed, as is the recently found North American-Asian relationship between Ictaluridae and Cranoglanididae.

Activation of FIP1L1-PDGFRα requires disruption of the juxtamembrane domain of PDGFRα and is FIP1L1-independent

Genetic abnormalities that result in expression of chimeric tyrosine kinase proteins such as BCR-ABL1 and ETV6-PDGFRbeta are common causes of hematopoietic malignancies. The paradigm for constitutive activation of these fusion tyrosine kinases is enforced homodimerization by self-association domains present in the fusion partner proteins. The unique interstitial deletion on chromosome 4q12 that leads to expression of the FIP1L1-PDGFRalpha fusion tyrosine kinase was recently identified as a cause of chronic eosinophilic leukemia. In this report, we demonstrate that FIP1L1 is completely dispensable for PDGFRalpha activation in vitro and in vivo. Instead, truncation of PDGFRalpha between two conserved tryptophan residues in the juxtamembrane (JM) domain is required for kinase activation and transforming potential of FIP1L1-PDGFRalpha. The presence of a complete JM domain in FIP1L1-PDGFRalpha is inhibitory, but this autoinhibition can be overcome by enforced homodimerization. Similar effects of the JM domain in the context of PDGFRbeta were observed. These results suggest that disruption of the autoinhibitory JM domain is an alternative, dimerization-independent mechanism by which chimeric tyrosine kinases are constitutively activated and induce leukemogenesis.