Werner Schempp

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9.

Overrepresentation of 3q and 8q material and loss of 18q material are recurrent findings in advanced human ovarian cancer

In order to define the ability of comparative genomic hybridization (CGH) to detect and map genetic imbalances, we investigated 47 malignant ovarian tumors and 2 ovarian tumors of low malignant potential. The most common genetic changes in order of frequency included DNA gains of chromosome arms 8q (53%), 3q (51%), 20q (43%), 1p (32%), 19q (30%), 1q (28%), 12p (28%), 6p (21%), and 2q (19%). The smallest regions of overrepresentation could be defined in 3q26‐qter, 8q23‐qter, 1p35‐pter, 12p12, and 6p21‐22, respectively. Losses were detected on 18q (23%), chromosome 4 (23%), 13q (17%), and 16q (17%) with the smallest underrepresented regions on 18q22‐qter, 13q21, and 16q23‐qter. Also, losses of the X chromosome (19%) were detected, correlating with higher ages of the patients. Therefore, some of these X chromosome losses might be due to a well‐known aging phenomenon and in these cases will be more preferably lost during cell division and tumor progression. Our findings show that ovarian carcinomas reveal consistent chromosomal abnormalities. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of oncogenes and/or tumor suppressor genes playing an important role in the tumorigenesis of ovarian carcinomas. Genes Chromosom Cancer 16:46–54 (1996).

Cytologic evidence for three human X-chromosomal segments escaping inactivation.

SummaryEarly replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).

Comparative mapping ofYRRM- andTSPY-related cosmids in man and hominoid apes

Using chromosomalin situ hybridization it has been demonstrated that specific members of theYRRM and theTSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with theYRRM-related cosmid A5F and theTSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and seconary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution ofYRRM andTSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.

Chromosome banding and DNA replication patterns in bird karyotypes

The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence.

Translocation breakpoints in three patients with campomelic dysplasia and autosomal sex reversal map more than 130 kb from SOX9

Campomelic dysplasia (CMPD1) and autosomal XY sex reversal (SRA1) are caused by mutations in the SRY-related gene SOX9 on 17q. Unexpectedly, the 17q breakpoints in four CMPD l translocation cases previously analyzed by us and others map 50 kb or more from SOX9. Here, we present clinical, cytogenetic, and molecular data from a new CMPD1/SRA1 patient with t(6; 17) (q14; q24). Fluorescence in situ hybridization has shown that the 17q breakpoint in this case maps to the same region as the breakpoints in the other translocation cases, at least 130 kb from SOX9. Likewise, the breakpoints in two of the previously described cases also map more than 130 kb and, as shown by pulsed field gel electrophoresis analysis, at most 400 kb or 690 kb from SOX9. By using a SOX9 coding sequence polymorphism, expression of both SOX9 alleles has been demonstrated by the reverse transcriptase polymerase chain reaction in lymphoblastoid cells from one of the translocation cases.

Centromere repositioning in mammals.

The evolutionary history of chromosomes can be tracked by the comparative hybridization of large panels of bacterial artificial chromosome clones. This approach has disclosed an unprecedented phenomenon: ‘centromere repositioning’, that is, the movement of the centromere along the chromosome without marker order variation. The occurrence of evolutionary new centromeres (ENCs) is relatively frequent. In macaque, for instance, 9 out of 20 autosomal centromeres are evolutionarily new; in donkey at least 5 such neocentromeres originated after divergence from the zebra, in less than 1 million years. Recently, orangutan chromosome 9, considered to be heterozygous for a complex rearrangement, was discovered to be an ENC. In humans, in addition to neocentromeres that arise in acentric fragments and result in clinical phenotypes, 8 centromere-repositioning events have been reported. These ‘real-time’ repositioned centromere-seeding events provide clues to ENC birth and progression. In the present paper, we provide a review of the centromere repositioning. We add new data on the population genetics of the ENC of the orangutan, and describe for the first time an ENC on the X chromosome of squirrel monkeys. Next-generation sequencing technologies have started an unprecedented, flourishing period of rapid whole-genome sequencing. In this context, it is worth noting that these technologies, uncoupled from cytogenetics, would miss all the biological data on evolutionary centromere repositioning. Therefore, we can anticipate that classical and molecular cytogenetics will continue to have a crucial role in the identification of centromere movements. Indeed, all ENCs and human neocentromeres were found following classical and molecular cytogenetic investigations.

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

SummaryTwo 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.

The Y-Chromosome Tree Bursts into Leaf: 13,000 High-Confidence SNPs Covering the Majority of Known Clades

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes.

Mapping chromosomal homology between humans and the black-handed spider monkey by fluorescence in situ hybridization

We hybridized human chromosome-specific DNA probes to metaphases of the New World monkey Ateles geoffroyito map the chromosomal homology between these two species. In the haploid Ateles geoffroyi karyotype the total number of signals was 51 for the 22 human autosomal probes used. Compared with Old World monkeys, the number of translocations found in the black-handed spider monkey karyotype was quite striking. The majority of these translocations are apparently Robertsonian and no reciprocal translocations were revealed. Nine autosomal human chromosome probes (11, 13, 14, 17, 18, 19, 20, 21, 22) provided only two signals each per metaphase, but six of these were translocated to subregions of different spider monkey chromosomes. The other 13 autosomal human chromosome paints (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16) provided fragmented signals. Three human probes (5, 8, 10) provided signals located on two pairs of spider monkey chromosomes. Four human paints (2, 3, 4, 12) provided hybridization signals on three pairs of chromosomes. Probes 6, 7, 15 provided six signals each on two pairs of chromosomes; probe 16 gave eight signals on two pairs of spider monkey chromosomes and probe 1 gave 12 signals on four pairs of chromosomes. The synteny between segments to human 18/8 appears to be an apomorphic ancestral condition for all New World monkeys. A synteny between regions homologous to human 16/10, 5/7 and 2/16 HSA is probably an apomorphic ancestral condition for all Cebidae. The syntenic association 3/15 and 4/1 is an apomorphic condition for the Atelinae.