47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation.

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.

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.

Heterogeneity of pericentric inversions of the human y chromosome.

Pericentric inversions of the human Y chromosome (inv(Y)) are the result of breakpoints in Yp and Yq. Whether these breakpoints occur recurrently on specific hotspots or appear at different locations along the repeat structure of the human Y chromosome is an open question. Employing FISH for a better definition and refinement of the inversion breakpoints in 9 cases of inv(Y) chromosomes, with seemingly unvarying metacentric appearance after banding analysis, unequivocally resulted in heterogeneity of the pericentric inversions of the human Y chromosome. While in all 9 inv(Y) cases the inversion breakpoints in the short arm fall in a gene-poor region of X-transposed sequences proximal to PAR1 and SRY in Yp11.2, there are clearly 3 different inversion breakpoints in the long arm. Inv(Y)-types I and II are familial cases showing inversion breakpoints that map in Yq11.23 or in Yq11.223, outside the ampliconic fertility gene cluster of DAZ and CDY in AZFc. Inv(Y)-type III shows an inversion breakpoint in Yq11.223 that splits the DAZ and CDY fertility gene-cluster in AZFc. This inversion type is representative of both familial cases and cases with spermatogenetic impairment. In a further familial case of inv(Y), with almost acrocentric morphology, the breakpoints are within the TSPY and RBMY repeat in Yp and within the heterochromatin in Yq. Therefore, the presence of specific inversion breakpoints leading to impaired fertility in certain inv(Y) cases remains an open question.

A non-human primate BAC resource to study interchromosomal segmental duplications.

Segmental duplications (SDs) are involved in the reshaping and evolutionary development of primate genome architecture. Their intrinsic property to promote genomic instability facilitates genome rearrangements, thereby contributing to karyotype diversity in primates. However, comparative analyses of SDs based on whole-genome shotgun assemblies of primate genomes may lead to a distorted view of their evolutionary dynamics as this method will incorrectly assemble or simply not represent these regions. Therefore high-quality sequences of chromosomally assigned SDs are indispensable for unraveling the amplification and dispersal pattern of SDs during primate evolution. Here, we use an updated version of the ancestral duplicon state of the non-palindromic SDs of all 4 human Y-chromosome euchromatin/heterochromatin transition regions to perform a survey of duplicons genome-wide across 7 primate species. By adjusting experimental conditions to the mean nucleotide sequence divergence to human we identified 11,075 BAC clones carrying primate orthologs or paralogs of human Y chromosome-derived duplicons. Preliminary results indicate lineage-specific amplification of duplicons in prosimians and gibbons. This BAC-based framework represents the first complete set of a defined number of duplicons over 60 million years of primate evolution. Comparative sequence analysis of this genetic resource can contribute to our deeper understanding of the impact of segmental duplications on primate genome evolution.

A family case of fertile human 45,X,psu dic(15;Y) males.

We report on a familial case including four male probands from three generations with a 45,X,psu dic(15;Y)(p11.2;q12) karyotype. 45,X is usually associated with a female phenotype and only rarely with maleness, due to translocation of small Y chromosomal fragments to autosomes. These male patients are commonly infertile because of missing azoospermia factor regions from the Y long arm. In our familial case we found a pseudodicentric translocation chromosome, that contains almost the entire chromosomes 15 and Y. The translocation took place in an unknown male ancestor of our probands and has no apparent effect on fertility and phenotype of the carrier. FISH analysis demonstrated the deletion of the pseudoautosomal region 2 (PAR2) from the Y chromosome and the loss of the nucleolus organizing region (NOR) from chromosome 15. The formation of the psu dic(15;Y) chromosome is a reciprocal event to the formation of the satellited Y chromosome (Yqs). Statistically, the formation of 45,X,psu dic(15;Y) (p11.2;q12) is as likely as the formation of Yqs. Nevertheless, it has not been described yet. This can be explained by the dicentricity of this translocation chromosome that usually leads to mitotic instability and meiotic imbalances. A second event, a stable inactivation of one of the two centromeres is obligatory to enable the transmission of the translocation chromosome and thus a stably reduced chromosome number from father to every son in this family.

Structural variation of the monoamine oxidase A gene promoter repeat polymorphism in nonhuman primates.

By conferring allele-specific transcriptional activity on the monoamine oxidase A (MAOA) gene in humans, length variation of a repetitive sequence [(variable number of tandem repeat (VNTR)] in the MAOA promoter influences a constellation of personality traits related to aggressive and antisocial behavior and increases the risk of neurodevelopmental and psychiatric disorders. Here, we have analyzed the presence and variability of this MAOA promoter repeat in several species of nonhuman primates. Sequence analysis of MAOA's transcriptional control region revealed the presence of the VNTR in chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), rhesus macaque (Macaca mulatta) and Gelada baboon (Theropithecus gelada). The majority of P. troglodytes and P. paniscus showed a single repeat with a sequence identical to the VNTR sequence in humans. In contrast, analyses of the remaining species revealed shorter sequences similar to the first 18 bp of human VNTR. Compared with other nonhuman primates, the VNTR sequence of M. mulatta showed the highest length variability with allele frequencies of 35, 25 and 40% for the five, six and seven repeat variants, respectively. The extent of variability of the MAOA promoter repeat in both rhesus monkeys and humans supports the notion that there may be a relationship between functional MAOA expression and aggression-related traits in humans and rhesus macaque populations.

Evolutionary breakpoint analysis on Y chromosomes of higher primates provides insight into human Y evolution.

Comparative FISH mapping of PAC clones covering almost 3 Mb of the human AZFa region in Yq11.21 to metaphases of human and great apes unravels breakpoints that were involved in species-specific Y chromosome evolution. An astonishing clustering of evolutionary breakpoints was detected in the very proximal region on the long arm of the human Y chromosome in Yq11.21. These breakpoints were involved in deletions, one specific for the human and another for the orang-utan Y chromosome, in a duplicative translocation/transposition specific for bonobo and chimpanzee Y chromosomes and in a pericentric inversion specific for the gorilla Y chromosome. In addition, our comparative results allow the deduction of a model for the human Y chromosome evolution.

The evolution of the azoospermia factor region AZFa in higher primates.

Clones of a PAC contig encompassing the human AZFa region in Yq11.21 were comparatively FISH mapped to great ape Y chromosomes. While the orthologous AZFa locus in the chimpanzee, the bonobo and the gorilla maps to the long arm of their Y chromosomes in Yq12.1-->q12.2, Yq13.1-->q13.2 and Yq11.2, respectively, it is found on the short arm of the orang-utan subspecies of Borneo and Sumatra, in Yp12.3 and Yp13.2, respectively. Regarding the order of PAC clones and genes within the AZFa region, no differences could be detected between apes and man, indicating a strong evolutionary stability of this non-recombining region.

Comparative mapping of human claudin-1 (CLDN1) in great apes.

The gene encoding claudin-1 (CLDN1) has been mapped to human chromosome 3 (HSA3; 3q28-->q29) using a radiation hybrid panel. Employing fluorescence in situ hybridization (FISH) we here show that a human P1-derived artificial chromosome (PAC) containing CLDN1 detects the orthologous sites in chromosomes of the great apes, chimpanzee, gorilla, and orangutan. Furthermore, the chromosomal position of CLDN1 was determined in mouse chromosomes by FISH. The position of fluorescent signals is confined to a single chromosomal site in both great apes and mouse and in each case maps to the chromosomal region that has conserved synteny with HSA3 (PTR2q28, GGO2q28, PPY2q38 and MMU16B1). Using a gene-specific probe our results are consistent with reports of the striking similarity of great ape and human genomes as illustrated previously by chromosome painting.

Chromosome painting shows that the proboscis monkey (Nasalis larvatus) has a derived karyotype and is phylogenetically nested within Asian Colobines.

The exceptional diploid number (2n=48) of the proboscis monkey (Nasalis larvatus) has played a pivotal role in phylogenies that view the proboscis monkey as the most primitive colobine, and a long-isolated genus of the group. In this report we used molecular cytogenetic methods to map the chromosomal homology of the proboscis monkey in order to test these hypotheses. Our results reveal that the N. larvatus karyotype is derived and is not primitive in respect to other colobines (2n=44) and most other Old World monkeys. The diploid number of 2n=48 can be best explained by derived fissions of a segment of human chromosomes 14 and 6. The fragmentation and association of human chromosomes 1 and 19 as seen in other Asian colobines, but not in African colobines, is best explained as a derived reciprocal translocation linking all Asian colobines. The alternating hybridization pattern between four segments homologous to human chromosomes 1 and 19 on N. larvatus chromosome 6 is the result of the reciprocal translocation followed by a pericentric inversion. N. larvatus shares this pericentric inversion with Trachypithecus, but not with Pygathrix. This inversion apparently links Nasalis and Trachypithecus after the divergence of Pygathrix. The karyological data support the view that Asian colobines, including N. larvatus, are monophyletic. They share many linking karyological features separating them from the African colobines. The hybridization pattern also suggests that Nasalis is nested within Asian Colobines and shares a period of common descent with other Asian colobines after the divergence of Pygathrix.

Cytogenetic mapping and orientation of the rhesus macaque MHC.

Applying fluorescence in situ hybridisation (FISH), six cosmid clones of rhesus macaque origin containing the genes SACM2L, RING1, BAT1 and MIC2, MIC3, MICD, and MOG of the major histocompatibility complex (MHC) were localised to the long arm of the rhesus macaque chromosome 6 in 6q24, the orthologous region to human 6p21.3. Furthermore, centromere to telomere orientation of the rhesus macaque MHC as well as the internal order of the MHC genes tested are the same as in human. Fiber-FISH allows a rough estimate of distances between these MHC genes in the rhesus macaque, and, as in the human, the rhesus macaque MHC comprises about 3 to 4 Mb.

Comparative mapping of CDY and DAZ in higher primates.

The human male specific expressed gene families CDY and DAZ are known to be repetitively clustered in the Y-specific region of the human Y chromosome. Comparative FISH-mapping of DNA clones specific for CDY and DAZ resulted in a Y-specific but diverse signal pattern within the non-recombining region of the Y-chromosomes of human and great apes. It can be concluded that the non-recombining part of the Y-chromosomes including CDY and DAZ, was exposed to species-specific amplifications, diversifications and rearrangements. Evolutionary fast fixation of any of these variations was possible as long as they did not interfere with male fertility.

The Azoospermia region AZFa: an evolutionar y view.

Compared to other regions on the human Y chromosome, the genomic segment encompassing the functionally defined AZFa locus has undergone higher X-Y sequence divergence, which is detectable by fluorescence in-situ hybridisation. This allows an evolutionary definition of an interval enclosing AZFa with a size of about 1.1 Mb. The region includes the genes USP9Y, DBY and UTY and is limited by evolutionary breakpoints within the PAC clones 41L06 and 46M11. These breakpoints restrict an area of possible male specific evolution that may have resulted in the acquisition of male specific functions, including a role in spermatogenesis.

Loss of the Y chromosomal PAR2-region in four familial cases of satellited Y chromosomes (Yqs).

Applying fluorescence in-situ hybridization (FISH) of various Y chromosomal DNA probes to four familial cases of human Yqs, it was possible to demonstrate that the formation of Yqs must have arisen from a reciprocal translocation involving the short arm of an acrocentric autosome and the heterochromatin of the long arm of the Y chromosome (Yqh). Breakpoints map within Yqh and the proximal short arm of an acrocentric autosome resulting in the gain of a nucleolus organizer region (NOR) including the telomere repeat (TTAGGG)n combined with the loss of the pseudoautosomal region 2 (PAR2) at the long arm of the recipient Y chromosome. In no case could the reciprocal product of an acrocentric autosome with loss of the NOR and gain of PAR2 be detected. Using the 15p-specific classical satellite-III probe D15Z1 in two of the four Yqs probands presented here, it could be shown that the satellited material originated from the short arm of chromosome 15. In contrast to the loss of PAR2 in Yqs chromosomes, another Y chromosomal variant (Yqh-) showing deletion of long-arm heterochromatin in Yq12 has retained PAR2 referring to an interstitial deletion of Yq heterochromatin in such deleted Y chromosomes.

Stable methylation patterns in interspecific antelope hybrids and the characterization and localization of a satellite fraction in the Alcelaphini and Hippotragini.

Conflicting data has recently appeared concerning altered methylation patterns in interspecific mammalian hybrids and the potential this may hold for driving karyotypic evolution. We report no detectable methylation difference in the genomic DNA of different interspecific F1 antelope hybrids (family Bovidae) and their parent species using the methylation-sensitive enzyme HpaII and its methylation insensitive isoschizomer MspI. However, both enzymes released a tandemly repeated satellite array. Characterization of the repeat using Southern blotting and a combination of sequencing, fluorescence in-situ hybridization (FISH) and C-banding, shows some similarity in the family of repeats between the hybridizing antelope species groups, and that the satellite is localized in the centromeric C-band positive regions of the chromosomes. Moreover, although there is little meaningful sequence homology with the well characterized bovine 1.715 satellite DNA, there is 86% sequence similarity with the sheep/goat satellite I, suggesting that they are related and are likely to have originated and evolved separately from the bovine unit.

Identification of two novel proteins that interact with germ-cell-specific RNA-binding proteins DAZ and DAZL1.

The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.

An SRY-negative 47,XXY mother and daughter.

Females with XY gonadal dysgenesis are sterile, due to degeneration of the initially present ovaries into nonfunctional streak gonads. Some of these sex-reversal cases can be attributed to mutation or deletion of the SRY gene. We now describe an SRY-deleted 47,XXY female who has one son and two daughters, and one of her daughters has the same 47,XXY karyotype. PCR and FISH analysis revealed that the mother carries a structurally altered Y chromosome that most likely resulted from an aberrant X-Y interchange between the closely related genomic regions surrounding the gene pair PRKX and PRKY on Xp22.3 and Yp11.2, respectively. As a consequence, Yp material, including SRY, has been replaced by terminal Xp sequences up to the PRKX gene. The fertility of the XXY mother can be attributed to the presence of the additional X chromosome that is missing in XY gonadal dysgenesis females. To our knowledge, this is the first human XXY female described who is fertile.

Familial mosaicism of del(Y) and inv del(Y).

Molecular cytogenetic investigation of a male proband showing oligozoospermia (OAT I-II degrees ) has led to the detection of a Y-chromosome mosaicism. This mosaicism consists of a deleted Y chromosome with deletion of most of the long-arm heterochromatin, including the PAR2, del(Y), and a Y chromosome, which, in addition to that deletion, shows a paracentric long-arm inversion, inv del(Y), with breakpoints in the DAZ gene cluster in deletion interval 6 and within the remainder of the long-arm heterochromatin of the Y. The Y mosaicism is not confined to the sterile proband but is also detected in both his father and his fertile brother. Interestingly, the percentage of inv del(Y) is highest (80%) in the proband showing oligozoospermia.

Transposition of SRY into the ancestral pseudoautosomal region creates a new pseudoautosomal boundary in a progenitor of simian primates.

We have isolated the prosimian lemur homologues for STS and SRY. FISH unambiguously co-localized STS with SHOX, IL3RA, ANT3 and PRK into the meiotic X-Y pairing region (PAR) of lemurs. In contrast to the close proximity of SRY to the pseudoautosomal boundary (PAB) on the Y chromosome in simian primates, SRY maps distant from the PAR in lemurs. Most interestingly, we were able to determine a DNA sequence divergence of 12.5% between the human and lemur SRY HMG box. This divergence directs to a 52 million year period of separate evolution of human and lemur SRY genes. Phylogenetically, this time period falls in between the times that prosimians and New World monkeys branched from the human lineage. Thus, we conclude that approximately 52 million years ago a transposition of SRY into the ancestral eutherian PAR distal to STS and PRK defined a new PAB in a simian progenitor. By this event, STS and PRK, amongst other genes, were excluded from the X-Y crossover process and thus became susceptible to rearrangements and/or deterioration on the Y chromosome in simian primates.

Mapping of members of the low-copy-number repetitive DNA sequence family chAB4 within the p arms of human acrocentric chromosomes: characterization of Robertsonian translocations.

Members of the long-range, low-copy-number repetitive DNA sequence family chAB4 are located on nine different human chromosome pairs and the Y chromosome, i.e. on the short arms of all the acrocentrics. To localize the chAB4 sequences more precisely on the acrocentrics, chAB4-specific probes together with rDNA and a number of satellite sequences were hybridized to metaphase chromosomes of normal probands and of carriers of Robertsonian translocations of the frequent types rob(13q14q) and rob(14q21q). The results demonstrate that chAB4 is located on both sides of the rDNA on all the acrocentrics; the exact location, however, may be chromosome specific. Chromosome 22, most probably, is the only chromosome where chAB4 is found in the direct neighbourhood of the centromere. Fluorescence in situ hybridization analyses of metaphase chromosomes of carriers of rob(21q22q) revealed breakpoint diversity for this rare type of Robertsonian translocation chromosome. A direct involvement of chAB4 sequences in recombination processes leading to the Robertsonian translocations analysed in this study can be excluded.