The first green lineage cdc25 dual-specificity phosphatase.

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

Identification of an autosomal recessive mode of inheritance in paediatric Behçet's families by segregation analysis.

We have conducted a segregation analysis in order to characterise the transmission of Behçet Disease (BD), a multifactorial condition with a strong genetic component. Complete information about BD status and pedigree was obtained on 104 probands from our database. We used the criteria of the International Study Group for BD (ISBD) to delineate the clinical status of the sibs: possible BD (patients meeting two criteria), or ascertained BD (patients meeting at least three criteria). A proband was defined as "paediatric" when he/she completed ISBD criteria before/by the age of 16 years. Families were distinguished as paediatric (n = 67) (ascertained through a paediatric proband), and non-paediatric (n = 37) ones. An Expectation Maximization (EM) algorithm was used to estimate the Mendelian segregation ratio P in nuclear families (two parents and their offspring). The maximum likelihood estimate: Pcirc; = 0.248, calculated in the paediatric data set, was consistent with the theoretical value of P = (1/4) for autosomal recessive inheritance, whereas the Pcirc; value was 0.08 when using the non-paediatric data set. Our work provides the first evidence of genetic heterogeneity in BD, and of the existence of a Mendelian entity in the paediatric BD subgroup. Previous studies failed to show any simple mode of inheritance in BD, probably because they were performed on the whole BD population.

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting.

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.

Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy.

We have developed a preimplantation genetic diagnosis (PGD) strategy for Duchenne muscular dystrophy (DMD) allowing the simultaneous amplification of four exons (6, 8, 28 and 32) of the dystrophin gene together with ZFX/ZFY genes for gender determination. Preliminary experiments were carried out on 215 single lymphocytes from male and female individuals. Amplification rates ranged from 90.2% for exon 6 to 96.7% for exons 8 and 32. At least four of the five sequences were successfully amplified in 95.8% of single cells, and sexing was possible in 98.5%. This 5-plex assay was found to be robust enough to be used in a PGD clinical procedure and was therefore applied to a family whose female partner was a heterozygous carrier of a large deletion extending from exon 21 to exon 34 of the dystrophin gene. We have thus analysed two exons located in the deleted region of the gene, two non-deleted exons used as intrasample controls, and ZFX/ZFY genes. Cleavage stage embryo biopsy followed by PCR resulted in transfer of three unaffected embryos. The advantage of the present approach is to identify and subsequently transfer unaffected male embryos in addition to female embryos, and is now applicable to all families displaying a deletion involving at least one of these exons.

First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers.

Retinoblastoma is a malignant intra-ocular tumour of developing retina initiated by inactivation of both alleles of the retinoblastoma susceptibility (RB1) gene. This paper reports the first clinical experience of preimplantation genetic diagnosis (PGD) for hereditary retinoblastoma using two highly polymorphic microsatellite markers RB1.20 and D13S284, located within and close to the RB1 gene respectively. Duplex PCRs were tested on more than 300 single lymphocytes from heterozygous individuals at both loci, in order to test the accuracy and reliability of the single-cell protocol. This procedure requires a nested PCR and the analysis of fluorescently labelled PCR products on an automatic DNA sequencer. Amplification efficiency and allele drop-out rates ranged from 96.7 to 98.4%, and 3.7 to 5.4% respectively. This test was found to be accurate and reliable enough to be applied to the study of human blastomeres. Subsequently, this approach was used in a PGD treatment cycle for a couple who already had a child affected with hereditary retinoblastoma and found to be informative for both microsatellite markers.

Multiplex fluorescent RT-PCR to quantify leukemic fusion transcripts.

The detection of chimeric transcripts derived from aberrant chromosomal fusion events provides an exceptionally valuable toolfor the diagnosis of leukemia. We have developed a simple, inexpensive, reproducible, and automated method to quantify RT-PCR products. Our approach utilizesfluorescent PCRfor the co-ampification of the specific fusion transcript with an internal control (HPRT). We have also combined the advantages of real-time quantitative PCR, namely continuous fluorescent detection of PCR products with the low cost of an endpoint assay by examining in a novel manner the amount offluorescent PCR product generated during the exponential phase of amplification. This has been achieved by using the automated loading and quantification capacity of a laser-induced fluorescence capillary electrophoresis system, the ABI PRIsMS 310A, so that we can effectively monitor amplification during the exponential phase cheaply, reproducibly, and in a sensitive manner. We have carefully verified our new technique using five leukemia cell lines, each expressing a differentfusion transcript. Specificity and reproducibility (cy within 10%) have been examined and demonstrate the excellent precision of our technology. The high sensitivity levels of at least 10(-4) to 10(-6) obtainedfor the serial dilutions of the five cell lines validate the choice of our fluorescent PCR as a comparable method to other more complicated and expensive methods. Our results have allowed us to quantify PCR products and the amount of chimeric mRNA originating from the translocation breakpoint. We demonstrate that our novelfluorescent method is useful to detect and quantify residual leukemic cells in patients undergoing therapy.

Enhanced cytokine mRNA levels in attack-free patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a recessively inherited inflammatory disorder, characterized by recurrent attacks of fever and serositis. Screening of mutations in the causing gene (MEFV) now allows accurate diagnosis of FMF among other inflammatory conditions. It is well documented that secreted levels of some pro-inflammatory cytokines are elevated in FMF. Here, we investigated cytokine expression at the transcriptional level, in patients that could be genetically ascertained. We have measured the transcript abundance of tumor necrosis factor alpha, interleukin-1beta, interleukin-6 and interleukin-8, in circulating leukocytes and shown that these were more elevated in attack-free FMF patients than in controls (P=0.01, P=0.008, P=0.02, P=0.001 respectively). There was no significant difference according to MEFV genotype or colchicine treatment. Our results suggest that cytokine transcriptional pathways are misregulated in attack-free FMF patients, and further supports the hypothesis that these patients have subclinical inflammation between attacks.

Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions.

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-coding genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F. rubripes. In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available, mouse sequences confirm conservation of the elements. From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.

The myosin light chain kinase gene is not duplicated in mouse: partial structure and chromosomal localization of Mylk.

The gene encoding myosin light chain kinase (MYLK) is duplicated on human chromosome 3 (HSA3; 3p13;3q21) and on a chromosome with conserved synteny to HSA3 in most non-human primate species. In human, the functional copy resides on 3q21, whereas the 3p13 site contains a pseudogene. To trace the origin of the duplication, we characterized the mouse gene Mylk. A single sequence corresponding to the functional Mylk was detected. We sequenced a 180-kb bacterial artificial chromosome clone containing the 24 first exons of Mylk; the complete mouse gene is expected to span >200 kb. Comparisons with the draft of the human genome revealed that the sequence and structure of MYLK are conserved in mammals. Fluorescence in situ hybridization (FISH) analysis indicated that the mouse gene localizes to a single site on chromosome 16B4-B5, a region with conserved synteny with HSA3q. Our study provides information on both the structure and the evolution of MYLK in mammals and suggests that it was duplicated after the divergence of rodents and primates.

Segregation of a mutation in CNGB1 encoding the beta-subunit of the rod cGMP-gated channel in a family with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal diseases leading to blindness. By performing full genome linkage analysis in a consanguineous French family affected with severe autosomal recessive RP, we have excluded linkage to known loci involved in RP and mapped a novel locus to chromosome 16q13-q21 (Zmax=2.83 at theta=0 at the D16S3089 locus). Two candidate genes KIFC3 and CNGB1 mapping to this critical interval have been screened for mutations. The CNGB1 gene, which encodes the beta-subunit of the rod cGMP-gated channel, is mutated in the family presented in this study.

From first base: the sequence of the tip of the X chromosome of Drosophila melanogaster, a comparison of two sequencing strategies.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.

The MICA region determines the first modifier locus in familial Mediterranean fever.

OBJECTIVE: Familial Mediterranean fever (FMF) is a genetically recessive inflammatory disease caused by mutations in the MEFV gene. Most patients of non-Ashkenazi Jewish ancestry or those who are homozygous for M694V manifest a severe disease course, but some express a mild form of the disease. We therefore searched for other genes which could possibly be implicated in the disease phenotype. We tested MICA (major histocompatibility complex class I chain-related gene A) because it has been associated with a number of other inflammatory disorders. METHODS: One hundred fifty FMF probands and their family members were evaluated. The MEFV gene was screened by a combination of denaturing gradient-gel electrophoresis, restriction fragment length polymorphism, and amplification refractory mutation system. The MICA transmembrane polymorphism in exon 5 was analyzed after biotin-labeled polymerase chain reaction products were loaded onto sequencing gels and subjected to autoradiography. RESULTS: The contribution of MICA to the FMF phenotype was confirmed after adjustment for the patient's ancestry and for the MEFV genotype. MEFV was individually the most important prognostic factor for the disease. However, the impact of M694V homozygosity on the age at disease onset (OR 2.3) was aggravated if patients also inherited MICA-A9 (OR 6.3). In contrast, the frequency of attacks was found to be dramatically reduced (OR 0.16) in patients with MICA-A4. CONCLUSION: We have identified the first FMF modifier locus, MICA. FMF is the first model of a Mendelian disease associated with MICA. These results clarify, at least partly, the inconsistent phenotype-MEFV correlation in FMF.

Evaluation of dHPLC for CX26 mutation screening in patients from southern France with sensorineural deafness.

The GJB2 gene (or CX26 for connexin 26) is one of the major genes causing nonsyndromic sensorineural hearing loss (NSSNHL). More than 50 sequence variations have been identified as polymorphisms or associated with autosomal or recessive forms of deafness. Though a major mutation, 35delG, is easily detectable by PCR digest; it is often present in the compound heterozygous state in our population in trans with recurrent, but less frequent, mutations. The CX26 gene is composed of a single coding exon that facilitates sequencing strategies. However, for mutation screening purposes, it is necessary to use high-throughput and cost-effective genotyping methods. Therefore, we have assessed denaturing high-performance liquid chromatography (dHPLC) in patients with known mutations in the CX26 gene. We conclude that dHPLC analysis is suitable for rapid and reliable scanning of the gene in deaf patients.

MEFV mutations in Behçet's disease.

Familial Mediterranean fever (FMF) and Behçet's disease (BD), both inflammatory diseases, are highly prevalent in the Middle Eastern and Mediterranean populations. FMF is a Mendelian autosomic recessive disease linked to MEFV, a gene of unknown function. BD in contrast is a polyfactorial disease associated with the major histocompatibility complex. Because FMF and BD have epidemiological similarities, we asked whether the FMF gene was implicated in BD. We screened for the common MEFV mutations a cohort of 114 chromosomes from definite BD patients [meeting the criteria of the International study group] and probable cases [meeting at least two of these criteria]. We screened in parallel an ethnically matched cohort of FMF and control chromosomes. The M694V, V726A and E148Q mutations tended to be more frequent in definite BD (2.6%, 2.6%, and 5.2%, respectively) than in controls (0%, 0%, and 2.2%). The P706 polymorphism was found in 10.5% of the probable BD chromosomes, but in only 1.6% of the controls (p=0.01). Because some MEFV mutations were more frequent in BD than in controls, we suggest that they may act as additional susceptibility factors in BD.

Genomic sequencing reveals the structure of the Kcnk6 and map3k11 genes and their close vicinity to the sipa1 gene on mouse chromosome 19.

In this report we present the analysis of two overlapping mouse cosmid clones that contain the entire Kcnk6, Map3k11 and Pcnxl3 genes, as well as part of the Sipa1 gene. The sequence and genomic organisation of the Kcnk6 and Map3k11 genes are described in detail. Sipa1 and Map3k11, which have independently been mapped with low resolution to the centromeric region of mouse chromosome 19, are shown here to lie close to each other and to the Kcnk6 gene, which has not previously been mapped. This gene cluster maps to the vicinity of the Dancer (Dc) mutation, which involves inner ear abnormalities and circling phenotypes. Since potassium channels have been implicated in deafness disorders, we have analysed the Kcnk6 gene, which encodes a two-P domain potassium channel, in the Dc mutant. No Dc-causing mutation in the Kcnk6 coding region could be identified. However, we detected a polymorphism in the Kcnk6 gene that leads to a C-terminal extension of the encoded protein by eight amino acids.

Evolution of the Gypsy endogenous retrovirus in the Drosophila melanogaster subgroup.

We conducted a phylogenetic survey of the endogenous retrovirus Gypsy in the eight species of the Drosophila melanogaster subgroup. A 362-bp fragment from the integrase gene (int) was amplified, cloned, and sequenced. Phylogenetic relationships of the elements isolated from independent clones were compared with the host phylogeny. Our results indicate that two main lineages of Gypsy exist in the melanogaster subgroup and that vertical and horizontal transmission have played a crucial role in the evolution of this insect endogenous retrovirus.

Familial Mediterranean fever in the 'Chuetas' of Mallorca: a question of Jewish origin or genetic heterogeneity.

Familial Mediterranean fever (FMF) is a hereditary disease commonly found among Jews, Armenians, Turks and Arabs. Recently, FMF was found in the 'Chuetas', a unique community on the island of Mallorca (Spain). To address the question of their possible Jewish origin, we analysed markers known to be linked to the gene responsible for FMF in Jews (MEFV) in this population. We found that 1/3 of the 16p13.3 chromosomes of the 'Chuetas' FMF patients bore the major ancestral haplotypes (S,S2) and their corresponding M694V and E148Q mutations, displayed by Jews from North Africa. Furthermore, we also detected a novel mutation (L110P) in this community. Yet 2/3 of these patients bore S negative haplotypes and lack the mutations commonly known to cause FMF. These results confirm that at least some of the 'Chuetas' share a common origin with Jews. However, they also provide evidence for the possibility of genetic heterogeneity in this disorder.