Small Duplications Research Articles

The genetic distance between humans and chimpanzees: What did Mary-Claire king and Allan Wilson really say in 1975?

Some scientific articles have been so often quoted and commented upon that scientists who feel they are fully conversant with their contents may actually be wrong. In 1975, King and Wilson proposed that the genetic distance between human beings and chimpanzees was less than 1% (King and Wilson 1975). Twenty-six years later, the wellknown paleoanthropologist Svante Paabo (2001) emphasized the impact that this article had, and the blow it was to our feeling of uniqueness and supremacy: chimpanzees and humans had been shown to be very close by the measure of the genetic distance between them. However, when the first draft of the chimpanzee genome was published in 2005, the facts turned out to be less simple (The Chimpanzee Sequencing and Analysis Consortium 2005). The famous 1% had become a myth (Cohen 2007) that had prevented a clear perception of the true differences. In fact, 1% represents the frequency of point mutations that occurred after the divergence of the human and chimpanzee lineages, but other genetic events, such as insertions and deletions of small DNA fragments and gene duplication, have to be taken into account to faithfully describe the genetic difference between humans and chimpanzees. What did King and Wilson really say? Reading their article is a shock: the interpretation that they gave for the 1% value differs greatly from that with which many, especially in the media, have become familiar. They considered, in fact, that the low genetic distance contrasts sharply with the huge morphological and behavioural transformations that occurred after the separation of the human and chimpanzee lineages. This means that measurement of the genetic distance is important when dating evolutionary events (see later), but not in explaining the major features in evolution. Evolution is the consequence of rare regulatory changes in a limited set of genes by chromosomal rearrangements. The article by Mary-Claire King and Allan Wilson is a mixture of what retrospectively appears as true and wrong statements and cannot be understood outside its scientific context.

Unusual Noncommunicating Isolated Enteric Duplication Cyst in Adults

Duplication cysts are rare gastrointestinal congenital abnormalities and can occur anywhere within the gastrointestinal tract. Duplication cysts are firmly attached to or share the wall of the alimentary tract and have a common blood supply with the adjacent segment of the bowel. Completely isolated duplication cysts are an extremely rare variety of gastrointestinal duplications with their own exclusive blood supply, and they do not communicate with the intestine. These cysts are usually diagnosed during early childhood, and very rarely detected in adults, mostly incidentally, due to a lack of symptoms. A 28-year-old male was admitted to our hospital with a chief complaint of lower abdominal pain and distention and a palpable mass for 1 month. Based upon computed tomography and sonographic findings, a small bowel duplication cyst was tentatively diagnosed. The cyst had no connection to the gastrointestinal tract. Herein we report the case of a noncommunicating isolated ileal duplication cyst in an adult. Resection of the cyst was performed safely without requiring bowel resection.

An Uncommon Location of Meckel's Diverticulum or Small Intestine Duplication? Case Report and Literature Review

Meckel's diverticulum is the most common congenital abnormality of alimentary tract. The antimesenteric location is one of the cardinal attribiutes of this pathology. We report case which tries to verify this dogma. The literature regarding uncommon location of Meckel's diverticulum was also reviewed.

Molecular characterization of an allelic series of mutations in the mouse Nox3 gene.

The inner ear consists of the cochlea (the organ of hearing) and the vestibular system (the organs of balance). Within the vestibular system, linear acceleration and gravity are detected by the saccule and utricle. Resting above the neurosensory epithelia of these organs are otoconia, minute proteinaceous and crystalline (calcite) inertial masses that shift under the physical forces imparted by linear movements and gravity. It is the transduction and sensation of these movements and their integration with vision and proprioceptive inputs that contribute to the sensation of balance. It has been proposed that a reactive oxygen species- (ROS-) generating NADPH oxidase comprising the gene products of the Nox3, Noxo1, and Cyba genes plays a critical and constructive role in the process of inner-ear development, specifically, the deposition of otoconia. Inactivation in mouse of any of the NADPH oxidase components encoded by the Nox3, Noxo1, or Cyba gene results in the complete congenital absence of otoconia and profound vestibular dysfunction. Here we describe our use of PCR, reverse transcription-PCR (RT-PCR), and rapid amplification of cDNA ends (RACE) with traditional and high-throughput (HTP) sequencing technologies to extend and complete the molecular characterization of an allelic series of seven mutations in the Nox3 gene. Collectively, the mutation spectrum includes an endogenous retrovirus insertion, two missense mutations, a splice donor mutation, a splice acceptor mutation, premature translational termination, and a small duplication. Together, these alleles provide tools to investigate the mechanisms of otoconial deposition over development, throughout aging, and in various disease states.

A Molecularly Defined Duplication Set for the X Chromosome of Drosophila melanogaster

We describe a molecularly defined duplication kit for the X chromosome of Drosophila melanogaster. A set of 408 overlapping P[acman] BAC clones was used to create small duplications (average length 88 kb) covering the 22-Mb sequenced portion of the chromosome. The BAC clones were inserted into an attP docking site on chromosome 3L using ΦC31 integrase, allowing direct comparison of different transgenes. The insertions complement 92% of the essential and viable mutations and deletions tested, demonstrating that almost all Drosophila genes are compact and that the current annotations of the genome are reasonably accurate. Moreover, almost all genes are tolerated at twice the normal dosage. Finally, we more precisely mapped two regions at which duplications cause diplo-lethality in males. This collection comprises the first molecularly defined duplication set to cover a whole chromosome in a multicellular organism. The work presented removes a long-standing barrier to genetic analysis of the Drosophila X chromosome, will greatly facilitate functional assays of X-linked genes in vivo, and provides a model for functional analyses of entire chromosomes in other species.

Genome-Wide Analysis of Genetic Alterations In Acute Myeloid Leukaemia (Massively parallel, high-throughput, paired-end DNA sequencing and genotyping of an AML genome).

AbstractAbstract 1690 Background –Acute Myeloid Leukaemia (AML) has a worldwide incidence of approximately 3.5 per 100,000 population per year with most cases occurring in adults. Survival at 12 months is less than 30% and at 5 years is less than 10% (NCI, 2010). AML is a complex disease that demonstrates marked heterogeneity morphologically, cytogenetically and molecularly. The largest cytogenetic subgroup of AML has normal cytogenetics and accounts for approximately 45% of de novo AML cases. Furthermore, the response to treatment and survival outcomes of cytogenetically normal AML (cn-AML) is remarkably heterogeneous. Aims –– To undertake whole-genome and exome sequence analysis of a cn-AML case at diagnosis and at relapse to identify subtle, potentially oncogenic mutations at the molecular level that may initiate AML and those that may be responsible for drug resistance or relapse. Specific Aims:i. –Comparison of the AML genomes at diagnosis, remission and relapse to identify genomic regions with changes in copy number and loss of heterozygosity, using single nucleotide polymorphism (SNP) microarray analysis. ii. Identify chromosomal rearrangements such as small insertions, duplications, inversions and deletions, (“indels”) and translocations that may contribute to AML initiation or relapse, using massively parallel, ultra-high throughput (“next generation”), paired-end sequencing of total genomic DNA at low depth. iii. Identify DNA point mutations potentially affecting protein function that may contribute to AML initiation or relapse by using next generation sequencing at high depth of the captured exomes (putative coding regions) of the AML and control genomes. Methods –The PwC (Price-Waterhouse-Coopers) Leukaemia and Lymphoma Tissue Bank, a joint initiative of the Australasian Leukaemia and Lymphoma Group and the Leukaemia Foundation, provided samples from an AML patient with normal cytogenetics and a blast count at diagnosis of 70% and at relapse of 85%. Genomic DNA extracted from autologous mesenchymal stem/stromal cells (MSCs) was used to represent non-leukaemic, germline, control DNA. Primary cell culture of MSCs from cryopreserved bone marrow aspirate cells (50 × 106) of our test patient at remission was achieved with standard tissue culture methods. High molecular weight DNA was extracted from the patient's MSCs and marrow cells at diagnosis, remission and relapse samples. Sonication (Covaris) and libraries appropriate for paired-end high-throughput sequencing (Illumina Genome Analyzer II instrument) were prepared from gel purified DNA fragments approximately 200 bp in size. Results and Discussion –Primary cell culture of MSCs from bone marrow aspirates proved to be a robust source of germline genomic DNA with several advantages over skin. Firstly, tissue banked samples, will not include biopsies of normal skin. Furthermore, skin can be contaminated by circulating leukaemic cells, which is problematic with low-depth genomic sequencing. The homogenous immunophenotype of the cultured MSCs indicate their purity. Preliminary SNP microarray analysis identified a large region of uniparental disomy (copy number neutral loss of heterozygosity) involving most of chromosome 13q, which was not identified by standard cytogenetic analysis. Low-pass or shallow paired-end genomic DNA sequencing has generated the following outputs. MSC genome: 10.7 Gb (3.6X haploid genome coverage) resulting from 152 × 106 paired sequence reads, AML_diagnosis genome: 21.6 Gb (7.2X) resulting from 142 × 106 paired sequence reads, AML_relapse genome: 26.8 Gb (8.9X) resulting from 177 × 106 paired sequence reads. The capture baits representing the human exome span 26,225,870 bp (approximately 0.9% of haploid human genome). Exome capture libraries were prepared from the three sources of DNA (MSC, AML_diagnosis and AML_relapse) and sequenced using a single lane for each library. The outputs for all three were very similar, approximately 23 × 106 paired sequence reads, representing 1.3 Gb (49X haploid exome coverage). The reads for whole genome or exome capture were of high quality and more than 98% could be unambiguously aligned to the human reference genome in the correct orientation and interval distance. Here we will present MSC enrichment results and sequencing output results, quality control and preliminary analysis of genomic alterations and exonic mutations. Disclosures:No relevant conflicts of interest to declare.

Micro-RNA copy number abnormalities in familial colorectal cancer

The majority of the familial colorectal cancer (CRC) cases cannot be explained by known gene defects, suggesting the existence of other genetic risk factors. In an approach to identify such risk factors, we recently performed a screen for copy number variations (CNVs) in high-risk CRC patients and found, among other lesions, several small deletions affecting microRNA genes. MicroRNAs are negative regulators of approximately 30% of the genes in the human genome, including numerous tumor suppressor genes and oncogenes. In order to comprehensively investigate copy number variation in microRNA genes, and to reveal whether such variations may affect CRC predisposition, we screened for CNVs of microRNA genes in 52 unexplained familial early-onset CRC patients using a custom-made ultimate (tiling) resolution oligo array containing 695 miR genes. We found various small (0.1e5 kb) constitutional deletions and duplications affecting single microRNA genes. Several of these (PCR-validated) CNVs could be detected also in normal controls, whereas others appeared to be rare and were thus far only found in CRC patients. We conclude that CNVs in microRNA genes are more common than previously thought, and that some of them may be associated with CRC predisposition. NOVEL TRANSLOCATION VARIANT IN EWING SARCOMA INVOLVING THE NFATC2 GENE

PORCN Mutations and Variants Identified in Patients with Focal Dermal Hypoplasia Through Diagnostic Gene Sequencing

Focal dermal hypoplasia (FDH) is an X-linked dominant disorder caused by mutations in the gene PORCN, which encodes a protein required for the secretion and signaling of Wnt proteins. While deletions are responsible for a small percentage of FDH-causing mutations, the vast majority of mutations are single-nucleotide substitutions or small deletions or insertions that can be identified by sequence analysis. In 2007, we implemented a PORCN gene sequencing test for individuals with a clinical diagnosis of FDH. To date, we have detected 12 novel PORCN mutations and 6 previously reported mutations in 53 such unrelated patients. The pathogenic PORCN mutations included nine nonsense mutations, three missense mutations, one small deletion, two small duplications, and three splice-site mutations. Of these mutations, two were found in affected men and were mosaic; one of these was found in three other affected women. The remaining 16 mutations were found only in women. All the mutations detected in women were presumed heterozygous. In addition to the disease-causing mutations, eight nucleotide variants of unknown significance were identified. Further characterization of these variants suggests that four of them are pathogenic mutations. These findings add to the heterogeneity of mutations in the PORCN gene that cause FDH.

High Resolution Array-CGH Characterization of Human Stem Cells Using a Stem Cell Focused Microarray

Human embryonic and induced pluripotent stem cells (ESCs, iPSCs) that are cultured for an extended period of time are susceptible to genomic instability. Chromosomal aberrations decrease the reliability and reproducibility of experiments and could deem the cells unusable for therapeutic purposes. The genetic stability of human ESCs and iPSCs is commonly monitored by karyotype analysis. However, this low-resolution technique can only identify large aneuploidies. A reliable, high-resolution technique to detect genomic aberrations at a cost comparable to karyotyping is needed to better characterize stem cell lines. We have designed a stem cell focused array-comparative genomic hybridization microarray that covers the entire genome at high resolution with increased probe coverage in over 60 stem cell associated genes and more than 195 cancer related genes. Several iPSC lines were analyzed using the focused microarray and compared with either karyotyping or a standard Agilent 44K microarray. In addition to the abnormalities detected by these platforms, the custom microarray identified several small duplications spanning stem cell and/or cancer related genes. Scientists using a stem cell focused microarray to characterize their stem cells will be aware of the structural variants present in their cells and be more confident in their experimental results.

Transcription Factor Mutations and Congenital Hypothyroidism: Systematic Genetic Screening of a Population-Based Cohort of Japanese Patients

Gene mutations of transcription factors that are predominantly expressed in the thyroid gland cause congenital hypothyroidism (CH). The prevalence of CH due to transcription factor mutations remains undetermined. This study was designed to define the prevalence of CH due to mutations of PAX8, NKX2-1 [encoding thyroid transcription factor (TTF)-1], FOXE1 (encoding TTF-2), and NKX2-5 among patients with permanent primary CH and in the general population in Japan. We enrolled 102 CH patients that represent 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. We sequenced PAX8, NKX2-1, FOXE1, and NKX2-5 using PCR-based methods. Additionally, deletion/duplication of PAX8, NKX2-1, and FOXE1 was screened by multiplex ligation-dependent probe amplification. Molecular functions of putative mutations were verified in vitro. We identified a novel small duplication of PAX8 (p.K80_A84dup) in two half-sibling patients with thyroid hypoplasia. We also found a novel NKX2-1 variation (p.H60W) in a sporadic nonsyndromic CH patient. In vitro experiments showed that K80_A84dup PAX8 had impaired transactivation of the thyroglobulin promoter. H60W TTF-1 exhibited a comparable transactivating capacity with wild-type TTF-1, suggesting a benign variation. We estimate the prevalence of PAX8 mutations to be 2.0% (two in 102) among Japanese CH patients and one in 176,000 (two in 353,000) in the general Japanese population. Using a population-based sample, we confirmed that a minor subset of CH patients has transcription factor mutations, but they are rare. In our cohort, PAX8 mutations were the leading cause of such a rare condition.

Improved structural characterization of chromosomal breakpoints using high resolution custom array-CGH

Array-CGH is a powerful tool for the rapid detection of genomic imbalances. By customizing the array it is possible to increase the resolution in a targeted genomic region of interest and determine the structure of the breakpoints with high accuracy, as well as to detect very small imbalances. We have used targeted custom arrays to zoom in on 38 chromosomal breakpoints from 12 different patients carrying both balanced and unbalanced rearrangements. We show that it is possible to characterize unbalanced breakpoints within 17-20,000 bp, depending on the structure of the genome. All of the deletion and duplication breakpoints were further refined and potential underlying molecular mechanisms of formation are discussed. In one of seven carriers of apparently balanced reciprocal translocations we detected a small deletion of 200 bp within the previously FISH-defined breakpoint, and in another patient, a large deletion of 11 Mb was identified on a chromosome not involved in the translocation. Targeted custom oligonucleotide arrays make it possible to perform fine mapping of breakpoints with a resolution within the breakpoint region much higher compared to commercially available array platforms. In addition, identification of small deletions or duplications in apparently balanced rearrangements may contribute to the identification of new disease causing genes.

Genomic responses to abnormal gene dosage: the X chromosome improved on a common strategy.

Mechanisms to guard genomic integrity are critical to ensuring the welfare and survival of an organism. Disruptions of genomic integrity can result in aneuploidy, a large-scale genomic imbalance caused by either extra or missing whole chromosomes (chromosomal aneuploidy) or chromosome segments (segmental aneuploidy). A change in dosage of a single gene may not compromise the well-being of an organism, but the combined altered dosage of many genes due to aneuploidy disturbs the overall balance of gene expression networks, resulting in decreased fitness and mortality [1],[2]. Chromosomal aneuploidy is a common cause of birth defects—Down syndrome is caused by an extra copy of Chromosome 21, and Turner syndrome by a single copy of the X chromosome in females. Furthermore, methods that detect segmental aneuploidy have uncovered small deletions or duplications of the genome in association with many disorders, such as mental retardation. Chromosomal and segmental aneuploidies are also frequent in cancer cells in which changes in copy number paradoxically increase cell fitness but are unfavorable to survival of the organism. A fundamental issue in biology and medicine is to understand the effects of aneuploidy on gene expression and the mechanisms that alleviate aneuploidy-induced imbalance of the genome. Chromosomal aneuploidy is caused by non-disjunction of chromosomes in meiosis or mitosis, while segmental aneuploidy involves breakage and ligation of DNA. In contrast, the sex chromosomes provide an example of a naturally occurring “aneuploidy” caused by the evolution of a specific set of chromosomes for sex determination that often differ in their copy number between males and females. For example, in mammals and in flies, females have two X chromosomes and males have one X chromosome and a Y chromosome, resulting in X monosomy in males. How does a cell or an organism respond to such different types of aneuploidy, abnormal or natural? It turns out that the overall expression level of a given gene is not necessarily in direct relation to the copy number. Unique strategies have evolved to deal with abnormal gene dosage to alleviate the effects of aneuploidy by dampening changes in expression levels. What's more, the X chromosome has evolved sophisticated mechanisms to achieve complete dosage compensation, not surprisingly, since the copy number difference between males and females has been evolving for a long time.

Four unrelated patients with lubs X‐linked mental retardation syndrome and different Xq28 duplications

The Lubs X-linked mental retardation syndrome (MRXSL) is caused by small interstitial duplications at distal Xq28 including the MECP2 gene. Here we report on four novel male patients with MRXSL and different Xq28 duplications delineated by microarray-based chromosome analysis. All mothers were healthy carriers of the duplications. Consistent with an earlier report [Bauters et al. (2008); Genome Res 18: 847-858], the distal breakpoints of all four Xq28 duplications were located in regions containing low-copy repeats (LCRs; J, K, and L groups), which may facilitate chromosome breakage and reunion events. The proximal breakpoint regions did not contain known LCRs. Interestingly, we identified apparent recurrent breakage sites in the proximal and distal breakpoint regions. Two of the four patients displayed more complex rearrangements. Patient 2 was endowed with a quadruplicated segment and a small triplication within the duplication, whereas patient 3 displayed two triplicated segments within the duplication, supporting that the Fork Stalling and Template Switching (FoSTeS) model may explain a subset of the structural rearrangements in Xq28. Clinically, muscular hypertonia and contractures of large joints may present a major problem in children with MRXSL. Because injection of botulinum toxin (BT-A; Botox) proved to be extremely helpful for patient 1, we recommend consideration of Botox treatment in other patients with MRXSL and severe joint contractures.

Wilms' Tumor 1 (WT1) Gene Mutations in Pediatric T-Acute Lymphoblastic Leukemia.

Abstract 3075Poster Board III-12The Wilms' tumor 1 (WT1) gene, located at chromosome band 11p13, encodes a transcriptional regulator involved in normal hematopoietic development. WT1 mutations have been identified in approximately 10 % of acute myeloid leukemia (AML), where it has recently been found to predict poor outcome, but also in T-cell acute lymphoblastic leukemias (T-ALL). Our aim was to evaluate the frequency, the main associated features and the prognostic significance of WT1 mutations in a cohort of pediatric patients with T-ALL treated according to EORTC-CLG trials. A total of 146 children, aged 7 months to 17 years, with newly diagnosed T-ALL were included in this study. Patients were treated according to 2 consecutive EORTC trials: 58 881 and 58 951. Immunophenotypic subtypes were classified according to the EGIL. Standard karyotype as well as molecular screening of HOX11/TLX1, HOX11L2/TLX3 and HOXA10 overexpression, SIL-TAL, NUP214-ABL, CALM-AF10 fusions were performed at diagnosis. WT1 transcript level was quantified by real-time PCR (RQ-PCR). Mutations of NOTCH1 exons 26, 27, 34, FBXW7 exons 9, 10, and WT1 exon 7, 9 were screened by direct sequencing. At least one WT1 mutation was found at diagnosis in 15 out of 146 (10%) T-ALL. WT1 mutations were predominantly exon 7 frameshift mutations (14/15 cases), consisting of small duplications, deletions or combined insertions/deletions, and were predicted to result in the production of a truncated protein missing the normal zinc finger domain. The remaining mutated patient harbored a somatically acquired missense mutation in exon 9 (C388Y), previously described in Denys-Drash syndrome. Only 4 out of 15 (27%) patients had 2 WT1 mutations and all WT1 mutations identified showed retention of the wild-type allele. Clonal evolution was investigated by analysis of 12 diagnostic-relapse pairs. Identical WT1 mutation was found at relapse in 3/4 mutated patients whereas 1/4 patients acquired an additional WT1 exon 7 mutation at relapse. One of the 8 patients with WT1 wild-type T-ALL at diagnosis acquired a WT1 exon 7 mutation at relapse. WT1 mutated and wild-type patients did not significantly differ in terms of age, gender, white blood cell count, or mediastinal involvement. Interestingly, WT1 mutated patients had significantly higher WT1 mRNA expression levels (median: 84% [25-837] for WT1 mutants vs 17% [0.007-657] for WT1 wild-type cases, p=0.005). This is in line with the trend for earlier developmental stage arrest observed in our WT1 mutated T-ALL as compared with WT1 wild-type T-ALL. Indeed, WT1 is preferentially expressed in immature hematopoietic progenitors and down-regulated in more differentiated cells. No association was found between the presence of WT1 mutations and NOTCH1 activating lesions. WT1 mutation was associated with HOX genes deregulation. HOX11 or HOX11L2 were overexpressed in 10/15 (67%) WT1 mutated ALL versus 29/123 (24%) WT1 wild-type ALL (p=0.001). In addition, HOXA overexpression and MLL-AF6 were found in one WT1 mutated T-ALL each. Overall, HOX deregulation was demonstrated in 12/15 (80%) WT1 mutated ALL at diagnosis and was also found in the T-ALL that acquired WT1 mutation at relapse. Despite being subclonal lesions strongly associated with HOX11 and HOX11L2 overexpression in T-ALL, WT1 mutations and NUP214-ABL fusion were found independent. A possible impact of WT1 mutation on outcome was investigated. The incidence of very high risk features was similar for patients with WT1 mutated and wild-type T-ALL. No significant differences were found between the WT1 mutated and wild-type group regarding 5-year event free survival (71.6% vs 74.1%; Wald test stratified for protocol: p=0.8) and overall survival (81.8% vs 81.3%; p=0.9). Notably, HOX112 overexpression, which is found in half of WT1 mutated T-ALL, has no pejorative impact either on outcome in EORTC trials. In conclusion, our study confirms that the type and incidence of WT1 mutations are very similar in pediatric T-ALL and AML, although the frequency of bi-allelic alterations may be lower in T-ALL. However, in contrast with AML, no pejorative outcome was associated with WT1 mutation. Moreover, we found that WT1 mutations are highly associated with direct or indirect aberrant HOX genes expression. DisclosuresNo relevant conflicts of interest to declare.

Developmental diseases and the hypothetical Master Development Program

Small deletions and duplications frequently occur in the pericentromeric region of chromosomes and many of these are associated with developmental abnormalities. These developmental syndromes are conventionally attributed to abnormal expression of protein-coding genes in the affected region. A hypothesis has recently been published concerning a Master Development Program based on noncoding transcripts from these regions (Parris GE. A hypothetical Master Development Program for multi-cellular organisms: Ontogeny and phylogeny. Biosci Hypotheses 2009;2:3-12.). This paper summarizes and expands the recently published hypothesis to include it application to developmental diseases. The author proposes that development of multi-cellular organisms is guided by a Master Development Program (MDP) located primarily in the pericentromeric heterochromatin. The MDP is believed to consist of a series of Generation-Specific Control Keys (GSCK) transcribed in sequence by Ikaros family transcription factors unless the GSCKs are suppressed by Sall1-family or Dnmt3b-family proteins. The MDP is proposed to increment with each cell cycle to the next GSCK resulting in development of the clone. A clone may be programmed to split into two clones as necessary through a two-cycle mitosis processes. The transcripts of the GSCKs presumably yield noncoding nuclear messenger RNAs (nmRNAs, 8-30 nt units) that act directly (e.g., as primers for RNA polymerase II) and indirectly to regulate HOX and other high-level transcription factor and developmental genes. As envisioned, the MDP would evolve by terminal addition of new GSCKs. The new GSCKs are produced by evolutionary consolidation of retro-transcripts into pyknons that collect and evolve at the end of the pericentromeric heterochromatin and are eventually incorporated into the MDP. The retro-transcripts are though to be produced during episodic retrovirus epidemics and account for punctuated equilibrium in species evolution.

A comparative approach shows differences in patterns of numt insertion during hominoid evolution.

Nuclear integrations of mitochondrial DNA (numts) are widespread among eukaryotes, although their prevalence differs greatly among taxa. Most knowledge of numt evolution comes from analyses of whole-genome sequences of single species or, more recently, from genomic comparisons across vast phylogenetic distances. Here we employ a comparative approach using human and chimpanzee genome sequence data to infer differences in the patterns and processes underlying numt integrations. We identified 66 numts that have integrated into the chimpanzee nuclear genome since the human-chimp divergence, which is significantly greater than the 37 numts observed in humans. By comparing these closely related species, we accurately reconstructed the preintegration target site sequence and deduced nucleotide changes associated with numt integration. From >100 species-specific numts, we quantified the frequency of small insertions, deletions, duplications, and instances of microhomology. Most human and chimpanzee numt integrations were accompanied by microhomology and short indels of the kind typically observed in the nonhomologous end-joining pathway of DNA double-strand break repair. Human-specific numts have integrated into regions with a significant deficit of transposable elements; however, the same was not seen in chimpanzees. From a separate data set, we also found evidence for an apparent increase in the rate of numt insertions in the last common ancestor of humans and the great apes using a polymerase chain reaction-based screen. Last, phylogenetic analyses indicate that mitochondrial-numt alignments must be at least 500 bp, and preferably >1 kb in length, to accurately reconstruct hominoid phylogeny and recover the correct point of numt insertion.

Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA

In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs.

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

BackgroundCytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family.ResultsA total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes.ConclusionThe characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

IFRD1 Is a Candidate Gene for SMNA on Chromosome 7q22-q23

We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia (SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the peripheral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as causative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation, animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological diseases.

Deleterious Variants of FIG4, a Phosphoinositide Phosphatase, in Patients with ALS

Mutations of the lipid phosphatase FIG4 that regulates PI(3,5)P(2) are responsible for the recessive peripheral-nerve disorder CMT4J. We now describe nonsynonymous variants of FIG4 in 2% (9/473) of patients with amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Heterozygosity for a deleterious allele of FIG4 appears to be a risk factor for ALS and PLS, extending the list of known ALS genes and increasing the clinical spectrum of FIG4-related diseases.