Giovanna Floridia

A Large AZFc Deletion Removes DAZ3/DAZ4 and Nearby Genes from Men in Y Haplogroup N

Deletion of the entire AZFc locus on the human Y chromosome leads to male infertility. The functional roles of the individual gene families mapped to AZFc are, however, still poorly understood, since the analysis of the region is complicated by its repeated structure. We have therefore used single-nucleotide variants (SNVs) across approximately 3 Mb of the AZFc sequence to identify 17 AZFc haplotypes and have examined them for deletion of individual AZFc gene copies. We found five individuals who lacked SNVs from a large segment of DNA containing the DAZ3/DAZ4 and BPY2.2/BPY2.3 gene doublets in distal AZFc. Southern blot analyses showed that the lack of these SNVs was due to deletion of the underlying DNA segment. Typing 118 binary Y markers showed that all five individuals belonged to Y haplogroup N, and 15 of 15 independently ascertained men in haplogroup N carried a similar deletion. Haplogroup N is known to be common and widespread in Europe and Asia, and there is no indication of reduced fertility in men with this Y chromosome. We therefore conclude that a common variant of the human Y chromosome lacks the DAZ3/DAZ4 and BPY2.2/BPY2.3 doublets in distal AZFc and thus that these genes cannot be required for male fertility; the gene content of the AZFc locus is likely to be genetically redundant. Furthermore, the observed deletions cannot be derived from the GenBank reference sequence by a single recombination event; an origin by homologous recombination from such a sequence organization must be preceded by an inversion event. These data confirm the expectation that the human Y chromosome sequence and gene complement may differ substantially between individuals and more variations are to be expected in different Y chromosomal haplogroups.

Transmission of a fully functional human neocentromere through three generations.

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.

Colocalization of (TTAGGG)n telomeric sequences and ribosomal genes in Atlantic eels.

The distribution of (TTAGGG)n telomeric repeats was studied in chromosomes of two Atlantic eels,Anguilla anguilla andA. rostrata. We found that these sequences hybridize to all the telomeres but also to the entire nucleolar organizer region (NOR) localized in both species at the short arm of chromosome 8. This was considered to be due to the interspersion of telomeric sequences within the NOR ones. Whatever the significance of this interspersion may be, it seems to be limited toA. anguilla andA. rostrata since inMuraena helena (family muraenidae), which also belongs to the Anguilliformes, no telomeric hybridization signals were found along the NOR regions.

Many Paths to the Top of the Mountain: Diverse Evolutionary Solutions to Centromere Structure

Do centromeres have a single evolutionary origin, or have eukaryotic segregation mechanisms evolved independently on more than one occasion? While centromeric DNA sequences can certainly appear de novo, as illustrated by neocentromeres, there are such striking similarities in the centromeric proteins (Figure 2Figure 2) that a single origin for the basic centromeric machinery, arising in an early eukaryotic ancestor, seems likely.How has the currently observed diversity arisen? S. cerevisiae centromeres should not be regarded as primordial, but as a special adaptation to the S. cerevisiae lifestyle. Perhaps the centromeres found in Drosophila, humans, and Arabidopsis are better models for a primitive centromere. If so, the recruitment of additional proteins, such as the CBF3 complex, might have converted an epigenetic mechanism into a sequence-specific one for S. cerevisiae. The evolutionary distribution of holocentric centromeres, which are found in organisms as diverse as plants and insects as well as Caenorhabditis, suggests that changes from one mode to the other have occurred on several occasions. Two recent studies of Drosophila show that these chromosomes can survive with more than one centromere. The dicentric chromosome C(1)A, carrying two copies of the Y centromere, has been maintained in Drosophila stocks for many years, and anti-Bub1 immunostaining shows that both centromeres can be active (Agudo et al. 2000xAgudo, M, Abad, J.P, Molina, I, Losada, A, Ripoll, P, and Villasante, A. Chromosoma. 2000; 109: 190–196Crossref | PubMedSee all ReferencesAgudo et al. 2000), while the bwD chromosome, which has been known for almost 60 years, shows two centromeres that stain with several anti-centromere/kinetochore antibodies including those against ZW10, and Cid (see Figure 2Figure 2) (Platero et al. 1999xPlatero, J.S, Ahmad, K, and Henikoff, S. Mol. Cell. 1999; 4: 995–1004Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesPlatero et al. 1999). When the distal heterochromatic block containing one centromere was detached in vivo using FLP-mediated recombination, centromeric activity including anaphase movement was seen. There are similarities between the protein components of holocentric and monocentric centromeres: Caenorhabditis HCP-3 (h olo c entric p rotein-3) is homologous to CENP-A, while two other holocentric proteins, HCP-1 and -2, are both homologous to mammalian centromeric protein CENP-F (Pidoux and Allshire 2000xPidoux, A.L and Allshire, R.C. Curr. Opin. Cell Biol. 2000; 12: 308–319Crossref | PubMed | Scopus (90)See all ReferencesPidoux and Allshire 2000). Perhaps a relatively minor change in the control of kinetochore size can convert a monocentric chromosome into a holocentric one.In conclusion, the centromere and kinetochore form the link between the highly conserved chromatin and microtubules. It is now apparent that there are many ways in which a basic set of ingredients, with specific modifications, can be used to make this connection. Genome sequencing projects have played an important part in revealing the molecular unity underlying the structural diversity of centromeres.‡To whom correspondence should be addressed (e-mail: chris@bioch.ox.ac.uk).

Mapping of a human centromere onto the DNA by topoisomerase II cleavage.

We have mapped the positions of topoisomerase II binding sites at the centromere of the human Y chromosome using etoposide‐mediated DNA cleavage. A single region of cleavage is seen at normal centromeres, spanning ∼50 kb within the centromeric alphoid array, but this pattern is abolished at two inactive centromeres. It therefore provides a marker for the position of the active centromere. Although the underlying centromeric DNA structure is variable, the position of the centromere measured in this way is fixed relative to the Yp edge of the array, and has retained the same position for >100 000 years.

Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq

A number of Xp22;Yq11 translocations involving the transposition of Yq material to the distal short arm of the X chromosome have been described. The reciprocal product, i.e. the derivative Y chromosome resulting from the translocation of a portion of Xp to Yq, has never been recovered. We searched for this reciprocal product by performing dosage analysis of Xp22-pter loci in 9 individuals carrying a non-fluorescent Y chromosome. In three mentally retarded and dysmorphic patients, dosage analysis indicated the duplication of Xp22 loci. Use of the highly polymorphic probe CRI-S232 demonstrated the inheritance of paternal Xp-specific alleles in the probands. In situ hybridization, performed in one case, confirmed that 29CL pseudoautosomal sequences were present, in addition to Xpter and Ypter, in the telomeric portion of Yq. To our knowledge, these are the first cases in which the translocation of Xp material to Yq has been demonstrated. The X and Y breakpoints were mapped in the three patients by dosage and deletion analysis. The X breakpoint falls, in the three cases, in a region of Xp22 that is not recognized as sharing sequence similarities with the Y chromosome, thus suggesting that these translocations are not the result of a homologous recombination event.

D8S7 is consistently deleted in inverted duplications of the short arm of chromosome 8 (inv dup 8p)

Ten patients with inverted duplication of 8p (inv dup 8p) were studied with cytogenetic, biochemical and molecular techniques. The duplication for the region 8p12-p22 was always associated with a deletion of the locus D8S7 (mapped in 8p23.1) as demonstrated with the probe pSW50 by both in situ hybridization and Southern blot. Restriction fragment length polymorphisms detected by probes pSW50 (1 case) and by pG2LPL35 (locus LPL) (two cases) were informative as to a maternal origin of the anomaly. The activity of glutathione reductase, whose gene maps in the duplicated region at 8p21.1, was increased in all patients. The recognizable phenotype of inv dup 8p includes neonatal hypotonia, prominent forehead, large mouth with everted lower lip, abnormally shaped large ears, brain malformations and severe mental retardation. Our findings indicate that the chromosome rearrangement is homogeneous at least for the presence of the deletion and support the hypothesis of a common mechanism of origin.

Quality assessment in cytogenetic and molecular genetic testing: the experience of the Italian Project on Standardisation and Quality Assurance.

Abstract The first Italian national trial of external quality assessment in genetic testing was organised within the framework of the “Italian National Project for Standardisation and Quality Assurance of Genetic Tests”. Sixty-eight Public Health Service laboratories volunteered for the trial, which involved molecular genetic tests (cystic fibrosis, β-thalassaemia, familial adenomatous polyposis coli and fragile-X syndrome) and cytogenetic tests (prenatal and postnatal, the latter included cancer cytogenetics). The response rate was high (88.2%). The level of analytical accuracy was good, i.e., the percentage of laboratories that correctly genotyped all samples was 89.3% for cystic fibrosis, 90.9% for β-thalassaemia, 100% for familial adenomatous polyposis coli (despite two laboratories did not complete the analysis because the amount of DNA was considered insufficient), and 90.5% for fragile-X syndrome. Written reports differed widely and were judged “inadequate” in over 50% of cases. Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries. Collaborative networks are strongly suggested to improve the quality of the reports.

Ataxic gait and mental retardation with absence of the paternal chromosome 8 and an idic(8)(p23.3): Imprinting effect or nullisomy for distal 8p genes?

Abstract A female child with mild dysmorphisms, motor and mental retardation had a 45,XX,-8,-8,+psu dic(8)(p23.3) karyotype in blood lymphocytes, skin fibroblasts and in a lymphoblastoid cell line. DNA analysis showed that the proposita was nullisomic for the 8pter region distal to D8S264, at less than 1 cM from the telomere. Analysis of DNA polymorphisms of 38 loci spread along the entire chromosome 8 revealed that only maternal alleles were present, distributed in four heterozygous and four homozygous regions. This finding indicated that the rearrangement occurred during maternal meiosis in a chromosome recombinant with a minimum of seven crossovers. To our knowledge this is the first case of uniparental maternal disomy for chromosome 8 and of nullisomy for the distal 1-cM portion of the short arm. The available data are in favour of the assumption that no imprinted genes are present on chromosome 8. Thus, dysmorphisms, motor and mental retardation of the proposita are likely to be caused by the nullisomy for the region distal to D8S264, a region in which a recessive gene for epilepsy with progressive mental retardation is known to be located.

Trisomy 8 syndrome owing to isodicentric 8p chromosomes: regional assignment of a presumptive gene involved in corpus callosum development.

Two patients with trisomy 8 syndrome owing to an isodicentric 8p;8p chromosome are described. Case 1 had a 46,XX/46,XX,-8,+idic(8)(p23) karyotype while case 2, a male, had the same abnormal karyotype without evidence of mosaicism. In situ hybridisation, performed in case 1, showed that the isochromosome was asymmetrical. Agenesis of the corpus callosum (ACC), which is a feature of trisomy 8 syndrome, was found in both patients. Although ACC is associated with aneuploidies for different chromosomes, a review of published reports indicates that, when associated with chromosome 8, this defect is the result of duplication of a gene located within 8p21-pter. Molecular analysis in one of our patients led us to exclude the distal 23 Mb of 8p from this ACC region.