EP0261172A1 - Y-specific dna hybridization probes and uses therefor - Google Patents

Abstract

The present invention relates to DNA specific for chromosome Y, which can be used as probes to establish unambiguously the presence or absence of regions of chromosome Y in the DNA of an individual, and to a method. of use of said probes. The DNA sequences of the Y chromosome can be used: in order to determine whether or not there is the presence of homologous sequences in a genome of an individual, in order to detect the presence or absence of specific parts of the Y chromosome in genomic DNA, and / or to determine the location of genes of interest on the Y chromosome.

Description

Y-SPECIFIC DNA HYBRIDIZATION PROBES AND USES THEREFOR

Description

Technical Field This invention is in the field of genetics and in particular relates to the analysis of genomes for the presence or absence of Y-specific DNA, particularly the testis-determining fragment(s).

Background Art In mammals, the primary sex-determining signal is the Y chromosome. Regardless of the number of X chromosomes per cell, mammalian embryos with a Y chromosome develop testes and those without a Y chromosome develop ovaries. XY, XXY, XXXY and XXXY embryos develop testes and XO, XX, XXX and XXXX embryos develop ovaries. The embryonic testes or ovaries establish a male or female hormonal environment, which determines the remainder of the sex phenotype, including the sex of the internal accessory organs and external genitalia. Wilson, J.D. et al., In; The Metabolic Basis of Inherited Disease (5th ed.), New York. Thus, the entire sex phenotype-- male or female -- is determined at the beginning by a gene or genes on the Y chromosome. This Y-borne gene or gene complex is referred to as the testis-determining factor (TDF).

Many structural anomalies of the human Y chromosome have been detected by light microscopy of stained mitotic chromsomes. Inferences as to the regional location of the testis determinant(s) on the human Y chromosome have been drawn from correlations of these abnormal karyotypes with the sex phenotypes. Buhler, E.M., Human Genetics, 55: 145-175 (1980); Davis, R.M., Journal of Medical

Genetics, 18: 161-195 (1981). Generally, assessment of chromosomal structural anomalies has made use of chromosome banding techniques, in which characteristic horizontal (density) differences (or bands) are detectable after the chromosomes have been treated (e.g., by staining with quinacrine or Giemsa; by controlled heat denaturation). Vogel, F. and A.G. Motulsky Human Genetics: Problems and Approaches. Springer-Verlag, pp 23-31 (1982). Normal males have a 46,XY karyotype; that is, there is a total of 46 chromosomes , including one X and one Y sex chromosome. Normal females are 46, XX, indicating a total of 46 chromosomes, including two X sex chromosomes. There are numerous anomalies associated with Y chromosome abnormalities. For example, XX males are sterile males with a 46,XX karyotype and testicular but no ovarian tissue; that is, the total number of chromosomes is 46 and both sex chromosomes are X chromosomes. About 1 in 20,000 males is an XX male.

In addition, there are human XY females with an apparently normal 46,XY karyotype. Such gonadal dysgenesis females have female external genitalia, uterus, fallopian tubes and streak ovaries. They have many features of the Turner syndrome, which is a condition usually associated with a 45, X chromosomal constitution. Many of the 46, XY females with the Turner phenotype are mosaic and have a 45, X cell line.

There are also very rare 45, X individuals who are sterile males with testes; it is presently unknown how maleness arises in these individuals.

The precision of chromosome banding techniques is limited, and, as a result, there is often considerable uncertainty as to the structure of abnormal Y chromosomes. For example, it has been hypothesized that XX males carry a small, male-determining portion of the Y chromosome, which cannot detected by conventional chromosome banding techniques. It has been shown, through the use of cloned Y sequences as DNA hybridization probes, that some XX males do have Y-specific DNA and that these males are heterogeneous with respect to the amount of Y DNA in their genes. Guellaen, G. et al., Nature, 307: 172-173 (1984); Page, D.C. et al., Nature, 315: 224-226 (1985). At this time, the testis determinant (s) has not been characterized and it is still unclear whether TDF maps to the short arm (Yp), the centromeriσ region, or long arm (Yq) of the Y chromosome.

The testis determinant (s) is not the only gene on the Y chromosome. However, genetic analysis of the mammalian Y chromosome has long been impeded by its haploid state. Unlike the other nuclear chromosomes, the Y has little opportunity to recombine with a homologue, making genetic linkage studies of the Y chromosome difficult, if not impossible. This at least partially accounts for the relative dearth of genes mapped to the Y chromosome in the mouse or human. Attempts to establish the Y-linkage of certain traits have been inconclusive because of the difficulty of distinguishing true Y-linked inheritance from sex-limited expression. Nonetheless, there is evidence for a number of genes on the Y chromosome in addition to the male determinant (s). For example, a structural gene for the antigen 12E7 has been shown to occur on the human Y chromosome. A structural or regulatory locus for the H-Y antigen probably maps to the Y chromosome in both the mouse and human. Two genes have been assigned to the weakly fluorescent proximal portion of Yq (band Yqll): a gene affecting spermatogenesis and a gene affecting height and tooth size. Their exact locations are unknown, however. At the present time, there is no satisfactory way of identifying and mapping genes of interest on the Y chromosome; of detecting anomalies of the Y chromosome or of correlating the presence or absence of specific regions of the chromosome with the determination of gonadal sex or with effects on these and other phenotypes.

Disclosure of the Invention

The invention described herein is based on the determination of the location on the normal Y chromosome of a large number of segments of human Y-chromosomal DNA; localization of the male (testis) determinant (s) on the Y chromosome; and construction of a physical or deletion map of the normal human Y chromosome. Genes located on the Y chromosome play an essential role in human male sexual development and the presence of the Y chromosome usually correlates with testis development. Conversely, Y chromosome abnormalities (e.g., deletions, rearrangements) are often associated with defects such as sexual dysfunction and mental retardation. Using presently available techniques, such as light microscopic analysis of stained chromosomes, it is not possible to make a definitive determination of the presence or absence of specific segments of the Y chromosome.

The deletion map of the human Y chromosome is composed of intervals which are defined by the portions of the Y chromosome shown, through hybridization with Y-DNA probes, to be present or absent in individuals with abnormal karyotypes or, as judged by cytogenetics, to have a structurally abnormal Y chromosome. The individuals whose genomic DNAs were tested included those with cytogenetically visible deletions of the long arm of the Y chromosome (Yq) and an apparently intact short arm of the Y chromosome (Yp) and those with cytogenetically detectable deletions of Yp and an apparently intact Yq. The hybridization results from these samples serve to orient the deletion map with respect to the long and the short arms of the chromosome.

The map serves as the basis for selection and cloning of Y-specific DNA sequences which are the subject of the present invention, as are methods for their use in analysis of sex chromosomal material. The map will be of great utility in clinical diagnosis and in the evaluation of any postulated Y function. With the map in hand, it is possible to systematically characterize structurally abnormal Y chromosomes by DNA hybridization even in cases where no abnormality can be detected by currently available techniques (e.g., chromosome banding). For example, the testis determining factor(s) (TDF) is mapped to the short arm of the Y chromosome (Yp). The cloned Y-specific sequences can be used, according to the method of the present invention, as DNA probes to establish unambiguously the presence or absence of regions of the normal Y chromosome in DNA from a subject. Using the deletion map of the normal Y chromsome, it is possible to systematically characterize Y chromsomes, particularly structurally abnormal Y chromosomes, by DNA hybridization, even in cases where chromosome banding has been unable to demonstrate an abnormality. The cloned Y-DNA sequences are useful as hybridization probes to analyze structural characteristics of the Y chromo- some; to determine whether Y-specific DNA is present in an individual's genome or not; to detect the presence or absence of specific portions of the Y chromosome in genomic DNA, as well as to determine the location of those portions or of the missing sequences on the Y DNA; and to diagnose genetic disorders and their related effects. In particular, one or more of the Y hybridization probes can be used to detect and characterize Y chromosome anomalies in individuals; to determine the occurrence in samples taken from patients of DNA sequences specific to the short arm (Yp), the centro meric region or the long arm (Yq) of the Y chromosome; to detect the region of the Y chromosome which is male-determining as well as the region determining the occurrence of spermatogenesis; and to detect other regions of interest (e.g., the region of the chromosome which apparently predisposes individuals with gonadal dysgenesis to gonadal neoplasms; the region responsible for expression of H-Y antigen). DNA prepared from blood or other tissue from a subject is analyzed by hybridization with one or more of the Y-specific probes and, as a result, it is possible to establish the presence or absence of specific regions of the Y chromosome which are of interest in, for example, a clinical or diagnostic context.

Brief Description of the Drawings

Figure 1 is the deletion map of the normal human Y chromosome constructed on the basis of DNA hybridization studies. Figure la is a deletion map in which an inversion polymorphism is represented.

Figure 2 is an autoradiogram showing results of hybridization of two labeled DNA probes to a gel transfer of Taql-digested DNAs from a normal female (case 28); an XX male (case 12); two XYq(-)males (cases 25 and 26) and a normal male (case 29).

Figure 3 is an autoradiogram showing results of hybridization of two labeled probes to gel transfers of Taql-digested DNAs from a normal male (case 29); a normal female (case 28) and an XX male (case 10). Figure 4 is a representation of the genetic deletion map of the human Y chromosome. Figure 5 is an autoradiogram showing results of hybridization of probe Y431-HinfA, which detects a Y-specific 2. lkb Hae III fragment, with Hae Ill-digested DNA from parents of a 45,X/46,XY male; a control female; and a 45,X/46,XY male.

Best Mode of Carrying Out the Invention

Most of the Y chromosome, which is the only haploid human chromosome, does not participate in meiotic recombination. As a result, it is impossible to construct a genetic map of the Y chromosome on the basis of recombinational distances among markers. A wide variety of deletions of the Y chromosome do, however, occur naturally. Attempts have been made to infer the regional location of the testis determining factor(s) (TDF) on the human Y chromosome from correlations of these abnormal karyotypes with the sex phenotypes of individuals having the abnormal karyotypes. The precision of these chromosome-banding studies is very limited, however, and there is considerable uncertainty as to the structure of abnormal Y chromosomes. Debate still continues as to the location of TDF. Until construction of the deletion map of the Y chromosome as described herein, it was not clear whether TDF maps to the short arm (Yp), the centromeric region or the long arm (Yq) of the Y chromosome.

The present invention relates to Y-chromosomal DNA sequences which occur on the normal Y chromosome and can be used as hybridization probes in theanalysis of DNA (i.e., sex chromosomes), prepared from blood or other tissue, for the occurrence of homologous DNA sequences. Through their use it is possible, in particular, to establish unambiguously in individuals, the presence or absence of Y DNA (i.e., DNA which occurs in normal Y chromosomes) and to determine the location of DNA sequences of interest on the Y chromosome. For example, using Y-specific DNA probes of the present invention, it is possible to detect the occurrence of the male-determining region of the Y chromosome, the centro- mere, and other regions of the sex chromosome which are of interest in a diagnostic or clinical context (e.g., the region thought to predispose to gonadal neoplasms in individuals with gonadal dysgenesis; the region which affects spermatogenesis; the region responsible for H-Y antigen expression). Such Y-specific probes can be used to analyze a set of chromosomes which appears cytogenically normal. A chromosome which appears on the basis of cytogenetic analysis, to be a normal X chromosome for the presence of homologous sequences, such as TDF. In addition, the Y-specific DNA probes can be used prenatally to assess sex chromosome structure; detection and identification of Y chromosome anomalies is thus possible. The following is a description of 1) the construction and characterization of the deletion map of the Y chromosome constructed on the basis of hybridization studies of genomic DNA from normal individuals and individuals having sex chromosome anomalies and 2) the use of Y-specific DNA as hybridization probes to detect DNA sequences homologous to those known to occur on the normal human Y chromsome.

I. A Deletion Map of DNA Sequences in the Y Chromosome A. Hybridization of DNAs from Normal Subjects and DNAs from Subjects Having Sex Chromosome Anomalies with Y-specific DNA Probes A physical or deletion map of the normal human Y chromosome was constructed by analysis and comparison of DNAs from normal subjects and subjects with anomalies of sex chromosomes. The map is represented in Figure 1.

Individuals were tested for the presence or absence of as many as 120 Y-DNA loci by hybridization to Southern transfers of restriction-digested genomic DNAs. Southern, E.M., Journal of Molecular Biology, 98: 503-517 (1975). The majority of individuals tested were XX males, XO males or XY females, or had, as judged by cytogenetics, a structurally abnormal Y chromosome. DNAs from normal male (46, XY) subjects and normal female (46, XX) subjects were also analyzed.

Most of the Y-DNA sequences used as hybridization probes were derived from a library made from flow-sorted Y chromosomes obtained from the National Laboratory Gene Library Project (Los Alamos). That is, the library consisted of a lambda phage (i.e., Charon 21A) into which fragments of Y chromosomal DNA (obtained by complete digestion of the DNA) had been cloned. Analysis of randomly selected Y-DNA-containing clones resulted in definition of deletion intervals on the chromosome. DNA sequences of interest were removed from the lambda phage and recloned into a plasmid (e.g., pUC8 or pUC13). Other DNA sequences used as probes are described by Page et al. in Nature, 311: 119-123 (1984) and by Bishop et al. in Journal of Molecular Biology, 173: 403-417 (1984); and also in Nature, 303: 831-832 (1983), the teachings of which are hereby incorporated by reference.

DNAs to be tested were prepared from peripheral leukocytes, cultured skin fibroblasts or EBV-transformed lymphoblasts according to published methods. Kunkel L. M. et al., Proceedings of the National Academy of Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289: 73-76 (1984). They were then digested with the restriction endonucleases TaqI or EcoRI and hybridized, as indicated above, using the method described by Southern, with selected radiolabeled DNA probes detecting Y-specific restriction fragments. B. Construction of the Deletion Map of the Y Chromosome Through Comparison of DNAs from Normal Subjects and Subjects of Abnormal Karyotype or Having A Structurally Abnormal Y

Chromosome DNA studies of 77 individuals having an abnormal karyotype or a structurally abnormal Y chromosome demonstrated that 47 of those tested carry part, but not all, of the Y chromosome. That is, in each of these 47 individuals, some, but not all, of the Y-specific restriction fragments invariably present in normal (46, XY) males were detected. Only 19 of these 47 Y deletions had been detected by cytogenetics; every Y deletion detected by cytogenetics was also revealed by DNA hybridization. The variety of patterns of Y-DNA loci occurring in these individuals are, as a group, most simply explained by the deletion map of the Y chromosome shown in Figure 1. This map accounts for each case on the basis of one Y breakpoint in all but two of the 47 individuals. That is, the deletion map of Figure 1 reconciles the hybridization data with the presence of a single, contiguous portion of the Y chromosome in all but two of the 47 cases.

The map has 10 deletion intervals (designated 1A through 7). Each of the intervals represented in Figure 1 is defined by the portion of the Y chromosome which is present or absent in a given individual or class of individuals or the difference between two individuals or two classes of individuals. For example, interval 1A is that portion of the Y chromosome present in the Class 1A XX male; the Class 1A XX male carries only one of the Y-specific fragments for which testing was done (and that fragment derives from or detects interval 1A). Interval 1B is that portion present in the Class 1B XX male minus the portion present in Class 1A XX males. That is, Class 1B XX males carry the same Y-specific fragment present in Class 1A XX males and an additional fragment, which derives from or detects interval B. Similarly, interval 2 is the portion of the Y chromosome present in the Class 2 XX males minus that portion present in Class 1B XX males. Class 2 XX males carry thirteen Y-specific fragments in addition to those carried by Class 1B XX males. Further studies using Y-specific DNA probes may indicate that one or more of these intervals should, in fact, be divided into two or more smaller intervals or subunits (e.g., as inter- val 1 has been divided into the two intervals designated as 1A and 1B). Interval 3, which is divided into two subunits, is the portion of the Y chromosome present in Class 3 XX males; such males have all those fragments present in Class 2 XX males plus 22 other fragments. Interval 4A is that portion of the Y chromosome present in Class 4 XX males but not in Class 3 XX males.

It is possible to orient the deletion map with respect to Yp and Yq as a result of data obtained from hybridization studies of individuals with cytogenetically detectable deletions of Yq and an apparently intact Yp (XYq(-)males) and individuals with cytogenetically detectable deletions of Yp and an apparently intact Yq (XYp(-)females). The centromere is assigned to Interval 4B on the basis of two observations. First, 4B is the only interval present in all deleted but independently segregating Y chromosomes. Second, it is known that a particular Y-specific repeated sequence localizes to the centromere by in situ hybridization. Wolfe, J. et al., Journal of Molecular Biology, 182:477-485 ( 1985) . This repeated sequence has now been mapped, using Y-specific DNA probles, to interval 4B. II. Use of Y-Specific DNA Probes in Explaining Sex Reversal Syndromes

There are several sex reversal syndromes in which it has been impossible to determine the genetic basis or explanation using traditionally available methods (e.g., chromosome banding studies). For example, there are individuals who have a 46,XX karyotype and are males; individuals who have a 46,XX karyotype and are hermaphrodites; individuals who have a 45,X karyotype and are males (designated XO males); and individuals who have an XY karyotype and are females.

Using Y-DNA specific probes and methods of detecting homologous DNA sequences according to thepresent invention, it is possible to determine the genetic basis for these apparent anomalies. A. XX Males For example, XX males are individuals who are sterile but are otherwise phenotypically males whose karyotype is 46, XX. That is, they have no Y chromosome and apparently (as judged by cytogenetics) have the chromosomes of a normal female. However, by definition, the gonads of XX males are comprised exclusively of testicular elements. Testes in such individuals might be due to the presence of male- determining chromosomal DNA which cannot be detected by traditional methods. Y-specific DNA probes were used to determine the occurrence of homologous sequences (i.e., Y-specific sequences) in genomic DNA of 46,XX males. By hybridizing as many as 120 different Y-DNA probes to Southern transfers, it was possible to detect certain Y-specific sequences in the genomes of 46, XX males. Based on the Y-specific sequences present, the XX males can be divided into six classes: Y(-) XX males, in whom no Y DNA sequences were detected, and five classes of Y(+) XX males, in whom Y DNA was detected.

The Y-specific DNA sequences present in the Y(+) XX males are those in intervals 1A-4A of Figure 1. Based on the hybridization studies using Y-specific DNA probes, these are assigned to Yp, the short arm of the Y chromosome. The studies showed that interval 1A was common to all Y(+) XX males, aswell as to ail XYq(-)males tested. As a result, if it is assumed that XX males have testes because of the presence of Y-derived TDF, TDF maps to interval 1. This assignment of TDF to Yp is in agreement with most karyotype-sex phenotype correlations. Y-specific DNA sequences have also been detected in one 47, XXX male tested using the probes and methods described herein. The portion of the Y chromosome present is similar, if not identical, to that present in Class 3 XX males. As mentioned, in one class of XX males (designated Y(-)), no Y DNA sequences have been detected to date, either through chromosome banding or through hybridization using Y-specific DNA probes. It is possible that Y(-) XX males carry portions of the Y chromosome even smaller than those found in the Class 1A Y(+) male. Alternatively, Y(-) XX maleness may be the result of an autosomal or X-chromosomal mutation.

B. XX Hermaphrodites XX hermaphrodites have a 46,XX karyotype and gonads which contain testicular as well as ovarian elements. Using Y-specific DNA probes, no Y-DNA sequences were detected in the genomes of three XX hermaphrodites tested. As with the Y(-) XX males, it is not known whether XX hermaphroditism is due to the presence of portions of the Y chromosome smaller than that in the Class 1A Y(+) male or possibly to an autosomal or X-chromosomal mutation. C. XO Males

"XO males" are sterile but otherwise phenotypiσ males with a 45,X karyotype. As judged by chromosome banding, they carry no part of the Y chromosome. XO males were examined using the battery of Y-DNA probes described above. DNA hybridization studies using these probes showed one XO male to be a low grade mosaic with an XY sex chromosome consti¬tution in less than 3% of fibroblasts. This mosaicism (later confirmed by karyotyping of many hundreds of cells) was detected and quantitated using probes detecting Y-specific repeated sequences. This analysis is described in detail in Example 4. Maleness is likely due to the XY cell line.

As judged by DNA hybridization, three other XO males carry intervals 1A through 4B of the Y chromosome (like Class 1 XYq(-)males). This is shown in Figure 1. That is, although banding studies by expert cytogeneticists had not detected any Y material, DNA hybridization showed that these three "XO" males actually carry the entire short arm and centromere of the Y in all or at least most of their cells. In two of these cases, in situ hybridization (i.e., hybridization of a radiolabelled DNA probe with metaphase stage chromosomes) and retrospective cytogenetics offer strong evidence that Y material has been translocated to an autosome, and in both cases, to 15p. In one case, the Y DNA probe attached (hybridized) to Yp (the short arm of the Y chromosome) even though no Y DNA had been demonstrated by pure cytogenetics. In these three cases, the male phenotype is explained by the presence of Y interval 1A. In a fifth XO male, no Y DNA has been detected, even in submolar amount. See Example 4. D. XY Females

Studies of XY females confirm the mapping of TDF to Y interval 1A. XY females have degenerate ovaries and no testicular tissue, despite the presence of. a Y chromosome. In most cases, XY females have similarly affected relatives, the inheritance being X-linked recessive (Swyer syndrome). In such cases, the defect is clearly on the X chromosome, and the Y is presumably intact. However, it seemed possible that some sporadic cases of XY femaleness might be due to deletions of TDF. By DNA hybridization, deletions of Y interval 1A (and variable numbers of adjoining intervals) were detected in four of nine sporadic XY females and in one female with a "balanced" Y;22 translocation. See Figure 1. In two of the XY females in whom deletions were detected by DNA hybridization, prometaphase banding studies had revealed small deletions in Yp. To the current limit of resolution, the deletions in the Class 1, 2 and 3 XY females equal the portions of the Y chromosome present in, respectively, the Class 3, 4 and 2 XX males. These findings suggest that XY females and XX males are reciprocal products of similar, aberrant X-Y exchanges.

Findings in XY females also shed light on the etiology of Turner syndrome, which most commonly occurs in females with a 45, X karyotype. All four of the XY females in whom deletions were detected had some signs of Turner syndrome (e.g., neck webbing, lymphedema); the five in whom deletions were not detected had no signs. These findings strongly suggest that Turner syndrome is the result of monosomy for a gene or genes common to the X and Y chromosomes. For example in XY females with Swyer syndrome, the X-linked recessive inheritance in familial cases suggests mutation in an X-chromosomal gene functioning in the pathway of gonadal differentiation, perhaps "downstream" of the Y-encoded TDF. The absence of Turner stigmata in these XY Swyer females argues that a normal Y chromosome is--like a second X chromsome-- capable of preventing the Turner phenotype. However, the presence of Turner stigmata in the other XY females--those with Yp deletions documented by DNA hybridization--strongly argues that the portion of Yp deleted in those females is required for the "Turner-blocking" effect. Turner syndrome has thus been shown to be due to monosomy for one or more genes common to the X and the Y chromosome. One or more of these genes can be mapped to the distal segment of Yp. III. Use of Y-Specific DNA Hybridization Probes to

Detect and Identify Homologous DNA Sequences of

Sex Chromosomes

Thus, as a result of the detection and identification of Y-specific DNA sequences and their regional localization on the Y chromosome, it is possible to use selected DNA sequences to analyze sex chromosomes prepared from blood or other body tissues for the occurrence of sequences homologous to the sequences selected. The selected DNA sequences are used as probes which detect the presence or absence of the homologous Y-DNA through hybridization with it.

For example, it is possible to use one or more probes which are well characterized and of known location on the normal Y chromosome to determine the presence or absence of homologous sequences in each of the intervals of the sex chromosomes of individuals having suspected or presumed anomalies or to determine the occurrence of specific fragments, such as the TDF or the gene known to affect spermatogenesis.

DNA to be analyzed can be from, for example, peripheral leukocytes, cultured skin fibroblasts or EBV-transformed lymphoblasts and can be prepared according to published methods. In one method, nuclei are first released by cell lysis in isotonic solution. Nuclei are then lysed in sodium dodecyl sulfate/proteinase K. DNA is purified by phenol extraction and dialysis. Kunkel, L.M. et al., Proceedings of the National Academy of Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289: 73-79 (1984).

The prepared DNA is subsequently subjected to restriction digestion, electrophoresis and transfer and is then hybridized with one or more Y-specific DNA probes, according to methods previously described by Page and de la Chapelle. See Page, D.C. and A. de la Chapelle, American Journal of Human Genetics, 36: 565-575 (1984), the teachings of which are incorporated by reference. The DNA can be digested, for example, with the restriction endonucleases TaqI or EcoRI. The resulting fragments are separated, for example, by agarose gel electrophoresis and transferred to membrane filters for hybridization to the Y-specific DNA sequences (probes). Hybridization can be carried out by Southern blot technique. Southern, E.M., Journal of Molecular Biology, 98: 503-517 (1975).

If the occurrence and identification of DNA sequences along the sex chromosome is to be carried out in an individual who is suspected, on the basis of cytogenetic analysis, of having a sex chromosome anomaly, a set of probes can be used. For example, the set of probes can include a probe for each of the intervals shown to occur on the normal Y chromosome. Use of this set of probes would make it possible to detect the absence of regions normally present in the Y chromosome; to detect the presence of regions not present in the normal sex chromosome; and to determine the location on the chromosome of regions of interest. One set of probes which can be used to determine the presence of the 10 intervals of the normal Y chromosome as represented in Figure 1 is as follows:

Interval

Detected Probe Characteristics

1A pDP307 0.9Kb Hind III Y fragment subcloned into Hind III site of pUC-13

Interval (continued) Detected Probe Characteristics 1B pDP132 3.5Kb Hind III Y fragment subcloned into Hind III site of pUC-13 2 pDP61 1.0Kb EcoRI/TaqI Y fragment from insert of pll5, subcloned into AccI and EcoRI sites of pUC-8

3A pDP125 4.6 Kb Hind III Y fragment subcloned into Hind III site of pUC-13

3B 50f2 1.1Kb EcoRI Y fragment subcloned into EcoRI site of pBR327.

4A pDP34 2.2Kb EcoRI Y fragment subcloned into EcoRI site of pDP322

4B pDP97 5.3Kb EcoRI Y fragment from cosmid Y97 subcloned into EcoRI site of pUC-13

5 12f 5.0Kb EcoRI Y fragment subcloned into EcoRI site of pBR322

6 50f2 1.1Kb EcoRI fragment subcloned into EcoRI site of pBR322 7 pY431-HinfA 0.8Kb Hinfl Y fragment cloned into PstI site of pBR322 Note: pDP307; pDP132; pDP125; pDP34 each detect zX specific and Y specific single copy DNA sequences; pDP34 is the same as DXYS1. Use of this set of probes, or an analogous set having one probe for each interval of the Y chromosome, makes it possible to assess the occurrence of each of the regions along the Y chromosome DNA, as well as to determine their location on the chromosome.

A subset of these probes can, of course, be used to assay prepared DNA for the occurrence of regions of particular interest. For example, a subset having the probes listed below can be used to determine the presence of DNA which occurs on the short arm of the normal Y chromosome. Interval

Detected Probe 1A pDP307

1B pDP132

2 pDP61 3A pDP125

3B 50f2

4A pDP34

Similarly, a subset of probes can be used to detect the occurrence of DNA shown to occur on the long arm of the normal Y chromosome.

As mentioned, DNA sequences can be used to detect the presence of the TDF. The probes used can be, for example, one or more sequences shown to be located on the normal Y chromosome and to be necessary for development of testes. In particular, in this case the probe can be Y-specific DNA present in interval 1A as represented in Figure 1. One such probe which can be used is pDP307 (described above). The DNA sequences used as probes can be cloned sequences derived from a Y-enriched lambda phage library (such as that available from the National Laboratory Gene Library Project) and subcloned into plasmids. Alternatively, synthetic DNA sequences can be used. The DNA probes must be labelled (e.g., radioaσtively, by chemical modification such as biotinylation) or otherwise modified in such a way that in a sample they can be detected, identified and/or quantitated. Comparison of data obtained in this manner with the deletion map of the normal Y chromosome represented in Figure 1 makes it possible to determine whether the DNA sequences homologous to the Y-specific probes (and thus TDF) are present in the sample DNA. IV. Use of Probes Recognizing Other Regions of

Interest on the Y Chromosome

As indicated previously, the deletion map makes it possible to evaluate any postulated Y chromosome function. For example, if the Y chromosome carries a gene for H-Y antigen (a transplantation antigen), using the deletion map and selected probes, it will be possible to map the gene to a deletion interval on the chromosome. It has been hypothesized that H-Y antigen is TDF and that H-Y is a genetic determinant of gonadal sex and/or a determinant of spermatogenesis. If it is found to have a role in spermatogenesis, for example, a probe specific to the Y region to which H-Y maps can be produced for use in diagnosis of male infertility.

As mentioned, XY (gonadal dysgenesis) females are sterile females with a 46,XY karyotype, female external genitalia, uterus, Fallopian tubes and "streak" ovaries. In addition, there are XY/XO females, who have Turner syndrome. Such individuals often have gonadoblastoma and it has been postulated that the Y chromosome, or some portion of it, has a role in predisposing such individuals to gonadoblastoma. The deletion map can be used to charac¬terize sex chromosomes in such individuals and detect the abnormality apparently responsible for this predisposition. Once that abnormality has been identified and the interval (s) characterized and localized, DNA probes can be produced and used toassess prepared DNA (e.g., from blood or other tissues) from subjects for the occurrence of homologous sequences. V. Homologous DNA Sequences on the Human X and Y

Chromosomes

DXYS1 and similar loci. It has long been thought that the mammalian X and Y chromosomes are partially homologous. Using DNA hybridization probes, Page et al. discovered DXYS1, a site of extensive single-copy DNA homology between the human X and Y chromosomes. A 4.5-kb segment of single-copy DNA from a human genomic library was hybridized to Southern transfers of human DNAs digested with the restriction enzyme Taql. This probe revealed TaqI restriction fragments 11, 12 and 15 kb long. Among more than 100 unrelated individuals, all males exhibited the 15-kb fragment in addition to one of the other fragments. Some females displayed both the 11- and 12-kb fragments, while others had only the 11- or 12-kb fragment, and none had the 15-kb fragment. These results suggested that the 15-kb Taql fragment derived from the Y chromosome, while the 11- and 12-kb fragments were X-linked alleles. Page, D.C. et al., Proceedings of the National Academy of Sciences, USA, 79: 5352-5356 (1982).

Subsequent studies confirmed these inferences. Hybridization of the 4.5-kb probe to Taql-digested DNAs from 48 members of a single family demonstrated Y-linked inheritance of the 15-kb fragment and X-linked inheritance of the 11- and 12-kb fragments. Hybridization of this probe to Taql-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) showed segregation of the 15-kb allele with the human Y chromosome and segregation of the 11- and 12-kb alleles with human X chromosomes. This was the first demonstration of single-copy sequence homology between the X and Y chromosomes of any mammal. This site of homology between the human X and Y chromosomes has been designated DXYS1. Page et al., in: Recombinant DNA Applications to Human Disease (Caskey C and White R, eds.) Cold Spring Harbor (1983)).

Deletion mapping studies using random Y-DNA loci suggest that as much as half or about 7000 kb, of Yp is highly homologous to Xq13-q21 (on the long arm); these are referred to here as DXYS1-like loci. All 22 loci mapping to Yp intervals 1A, 1B, 2, 3A and 4A appear to have this origin. DXYS1 is the most thoroughly studied member of this class of X-Y homologous loci. If all of the DXYS1 loci mapped to the five different intervals on Yp are the result of a single transposition from the X at some time in the last few million years of human evolution (as studies suggest) then one might expect the homologous loci to occur tightly clustered, and in the same order, on the X chromosome. Most, if not all of these, DXYSl-like loci are found in the vicinity of Xq13-q21, as shown by physical mapping by Southern hybridization, using genomic DNAs from individuals or human-rodent hybrid cells carrying known portions of the X chromosome.

This physical map of DXYS1-like loci on the X can be complemented by a genetic linkage map among such loci. Eight X-linked RFLPs at DXYSl-like loci have been characterized. By simultaneously tracing the inheritance of several such X-linked RFLPs in families, it is possible to construct a genetic linkage map among these Y-homologous sites on the X chromosome.

X-linkage studies (i.e., to linkage map these DXYSl-like loci with respect to each other) simultaneously map the loci within the larger context of the X chromosome, in effect, generating a "fine-structure" physical/genetic linkage map in the vicinity of Xq13-q21.

Genetic mapping of these X-linked RFLPs at DXYSl-like loci tremendously enhances their usefulness in studies of X-linked traits. For example, more than 50 laboratories around the world are already using the X-linked RFLP at DXYS1 to investigate choroideremia Charcot-Marie-Tooth disease and other X-linked diseases. DXYS1 also appears to be closely linked to ectodermal dysplasia.

Pseudoautosomal Loci on the X and Y Chromosomes

Xp-Yp pairing during male meiosis has long been taken as evidence of homology and even recombination between those regions. Two loci that provide concrete evidence of this previously hypothetical region have been identified in the course of analyzing about 120 Y-DNA loci. Both loci are common to the X and Y chromosomes and exhibit frequent X-Y recombination as a normal event during male meiosis. They have been physically mapped to distal Xp and distal Yp, respectively. Because of X-Y recombination, restriction polymorphisms (RFLP) at these loci do not show strictly sex-linked inheritance, but instead are inherited as though autosomal; hence the term "pseudoautosomal." One of the pseudoautosomal probes detects a closely related family of sequences displaying a very high degree of RFLP. Most of this polymorphism can be detected using a number of restriction enzymes, so it is likely not due to base-pair substitutions. Not only the lengths but the numbers of homologous restriction fragments vary from individual to individual. Nonetheless, family studies suggest that this probe is detecting a single genetic locus. Within a family, it is possible to recognize a particular collection of autoradiographic bands as constituting an allele. Although this RFLP does not show sex-linked inheritance, the locus has been mapped to the distal short arms of the X and Y by in situ hybridization and deletion mapping in rodent-human hybrids. These and other pseudoautosomal loci probably map distal to Interval 1A on the short arm of the Y chromosome. VI. Identification of Y-linked RFLPs

Six Y-linked RFLPs have been characterized including one occurring at DXYS1, a site of extensive single-copy DNA homology between the human X and Y chromosomes. In most unrelated American white males, the Y chromosome carries a tandem duplication of 15 kb of this X-homologous sequence. The same is true of most Oriental and aboriginal Australian males. Thus, like the X-to-Y transposition that gave rise to the DXYS1 homology itself, this Y-linked RFLP appears to have arisen prior to the formation of the human races. These Y-linked RFLPs also may be useful in the refinement of the deletion map of Figure 1 and in determining whether the map is, in fact, dimorphic. That is, it may be instrumental in determining whether, among the population, there are two Y chromosomes which differ by an inversion of the intervals represented in Figure 1, particularly intervals 3B and 4A. For example, the map shown in Figure 1 does not account economically for the class 4XX male or the class 2XY female; in either case, three breakpoints would be required on the Y chromosome. An inversion polymorphism in which the order of intervals 3B and 4A are reversed accounts for each of these cases via one breakpoint on the Y and is consistent with the idea that XX males have a terminal portion of Yp. Class 3 XX males and class 1 XY females can be explained as arising from a Y chromosome having a 3A-3B-4A-4B sequence; the class 4 XX male and class 2 XY female can be explained as arising from a Y chromosome having a 3A-4A-3B-4B sequence.

The present invention will be further illus¬trated by the following examples, which are not intended to be limiting in any way. Example 1 Construction of Deletion Map of the Normal Y Chromosome Patients Studied DNAs from normal individuals and individuals having abnormal karyotypes or cytogenetically determined sex chromosome anomalies were hybridized with Y-specific DNA probes. DNAs obtained from 77 individuals were tested for the presence of as many as 120 Y-DNA loci. The majority of individuals tested were XX males, XO males or XY females or had, as judged by cytogenetics, a structurally abnormal Y chromosome.

The patients studies are listed in Table 1, which lists the phenotype and karyotype of each individual. Most of the individuals studied were karyotyped during the course of a medical evaluation because of infertility and/or small testes.

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DNA Extraction and Gel-Transfer Hybridization

DNA was prepared from peripheral leukocytes, cultured skin fibroblasts, or EBV-transformed lymphoblasts by published methods. Kunkel, L.M. et al., Proceedings of the National Academy of

Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289; 73-76 (1984). Restriction digestion, electrophoresis, transfer and hybridization of DNA were performed as previously described. Page, D.C. and A. de la Chapelle, American Journal of Human Genetics, 36: 565-575 (1984). Each hybridization probe was used at either "reduced" or "high" stringency, as described below. "Reduced" stringency implies that hybridizations were carried out at 42°C and that the final wash was in 2X SSC at 68ºC or in 0.1X SSC at 55ºC. "High" stringency implies that hybridizations were carried out at 47 °C and/or that the final wash was in 0.1X SSC at 68°C. DNA Hybridization Probes Used

With the exception of probes pDP34 and P1, all probes described below are plasmid subclones derived from a Y-enriched lambda phage library (see above). Bishop, C. et al., Journal of Molecular Biology, 173; 403-417 (1984); Bishop, CE. et al., Nature, 303 : 831-832 (1983). For many of the probes, the names of the homologous DNA segments or loci (e.g., DXYS5, DYZ1) are designated as assigned at the Human Gene Mapping Conference VII. Skolnick, M.H. et al., Report of the Committee on human gene. mapping by recombinant DNA techniques. Cytogenetics and Cell Genetics, 1-4; 210-273 (1984). Probes 47a and 47z have been shown to detect highly homologous sequences on the X and Y chromosomes (DXYS5). Geldwerth, D. et al., EMBO Journal, 1739:1743 (1985). At high stringency, 47a detects a Y-specific Taql fragment of 4.3 kb; 47z detects a Y-specific Taql frament of 3 kb. These sites of X-Y homology are also detected by probe 47c. Guellaen, G. et al., Nature, 307: 172-173 (1984). 47a, 47c and 47z are subclones from the same cosmid. Probe 13d has been shown to detect highly homologous sequences on the X and Y chromosomes (DXYS7). Geldwerth, D. et al., EMBO Journal, 1739:1743 (1985). At high stringency, 13d detects a Y-specific Taql fragment of 7 kb. Probe 115 detects highly homologous sequences on the X and Y chromosomes (DXYS8). Geldwerth, D. et al., EMBO Journal, 4:1739-1743 (1985). At high stringency, 115 detects a Y-specific Taql fragment of 2.1 kb. Probe 52d detects multiple loci on the Y chromosome as well as one on the X. Bishop, C. et al., Journal of Molecular Biology, 173 : 403-317 (1984). At reduced stringency, 52d detects Y-specific EcoRI fragments of 7 kb (restriction fragment 52d/A), 1.2 kb (52d/B), and 1.0 kb (52d/C; apparently an unresolved doublet). The corresponding Y-specific Taql fragments are 9 kb (52d/C; an unresolved doublet), 5 kb (52d/A), and 3 kb (52d/B). Probe 50f2 defines multiple Y-specific loci and an autosomal locus. At reduced stringency, 50f2 detects Y-specific EcoRI fragments of 10 kb(50f2/A), 7.5 kb (50f2/B), 6 kb (50f2/C), 4.5 kb (50f2/D) and 1.7 kb (50f2/E). The corresponding Y-specific Taql fragments are 9 kb (50f2/E), 8 kb (50f2/D), 3.5 kb (50f2/A or 50f2/B), and 3 kb (an unresolved doublet, corresponding to 50f2/C and either 50f2/A or 50f2/B). Guellan, G. et al., Nature, 307: 172-173 (1984).

Probe 118 detects numerous Y-specific restriction fragments. Four Taql fragments whose presence or absence could be unambiguously determined were considered in this study: 7 kb (118/A), 6 kb (118/B), 5 kb (118/C), and 1 kb (118/D). Guellan, G. et al., Nature, 307: 172-173 (1984).

Probe pDP34 detects highly homologous sequences on the X and Y chromosomes (DXYS1). Page, D.C. et al., Proceedings of the National Academy of Science, U.S.A., 79: 2352-2356 (1982); Page, D.C. et al., Nature, 311: 119-123 (1984). At high stringency, pDP34 detects a Y-specific Taql fragment of 15 kb. Probe 64a7 detects homologous sequences on the Y and on an autosome. Guellaen, G. et al., Nature, 307: 172-173 (1984). At reduced stringency, 64a7 detects a Y-specific Taql fragment of 4.5 kb.

Probe 12f detects sequences on autosomes and the X as well as on the Y. Bishop, C. et al.,

Journal of Molecular Biology, 173: 403-317 (1984). At high stringency, 12f detects two or three Y-specific Taql or EcoRI fragments. Samples were scored for the presence or absence of. an 8-kb, Y-specific Taql fragment (corresponding to a 5-kb EcoRI fragment). At reduced stringency, probe 49f detects numerous Y-specific fragments. Bishop, C. et al., Journal of Molecular Biology, 173: 403-317 (1984). Samples were scored only for the most intensely hybridizing Y-specific fragments (2.0- and 1.8-kb Taql, or 2.8-kb EcoRI).

DYZ1 repeats were probed with a mixture of several cloned 3.4-kb Haelll repeated elements. DYZ1 repeats have been detected at high stringency as a Y-specific EcoRI fragment of 3.4 kb or as several discrete Y-specific Taql fragments of less than 0.6 kb.

DYZ2 repeats were examined using probe P1, a subfragment of the 2.1-kb Haelll repeated element, Vergnaud, G. et al., British Medical Journal, 289: 73-76 (1984), or probe pY431-HinfA, Bernheim, A. et al., Proceedings of the National Academy of Sciences, U.S.A., 80: 7571-7575 (1983). DYZ2 repeats were characterized at high stringency as Y-specific smears of high molecular weight in both EcoRI and Taql digests. In addition, discrete Y-specific fragments were observed in Taql digests. The results of hybridization of DNAs from the subjects with Y-specific DNA probes are best described with reference to the figures. Figures 2 and 3 are autoradiograms from representative hybridization experiments. For each probe used it was possible to score for unambiguously Y-specific bands. The diversity of probe types is illustrated in Figures 2 and 3; these probes are described in greater detail in Example 1. In Figure 2 (left panel) is shown a probe (49f) which detects autosomal as well as multiple Y-specific bands, some of which are polymorphic. In Figure 2 (right panel) is shown a probe (12f) which detects many autosomal and X-chromosomal bands which are present in all the individuals tested. It was also possible to score unambiguously for a Y-specific 8-kb Taql fragment.

In Figure 3 are shown two probes which each detect single-copy sequences on the X and Y chromosomes. They were hybridized to gel transfers of Taql-digested DNAs from a normal (46,XY) male, a normal (46, XX) females and an XX male. As shown, each probe detects X-chromosomal fragments, as well as a Y-specific fragment. In the case of probe 47a, the Y-specific fragment is 3kb and in the case of probe 47, it is 4.3kb.

The individuals tested were scored for the presence or absence of restriction fragments observed in normal males but not in normal females, and therefore assumed to derive from the Y chromosome. The restriction fragments detected are summarized in Table 1 (above). Some probes (e.g. 50f2, 118, and 52d) detect multiple Y-specific fragments. For such probes, individuals were scored for the presence or absence of the particular Y-specific fragments indicated in Table 1. As indicated in Table 1, no Y-specific DNA sequences were detected in XX hermaphrodites and XX males, referred to as Y(-) XX males).

The other individuals included XX males (referred to as Y(+) XX males); XYq()-males; and a t(Y;15) female. In each of these individuals, the presence of one or more Y-specific restriction fragments was detected. However, in none of these individuals were all the Y sequences for which testing was done detected. All of the Y sequences were present in each of the normal male controls and, when tested, in the fathers of the cases. Without exception, the Taql or EcoRI restriction fragments detected in the individuals were of the same size as those seen in normal males. This was also true in fathers of the cases who were tested. This leads to the conclusion that each of these cases contains a portion, but not all, of the Y chromosome. Based on a comparison of the Y-specific fragments detected (using the DNA probes described) in normal subjects and in subjects having chromosome anomalies, a deletion map of the Y-specific DNA sequences in the normal Y chromosome was constructed.

The data obtained are consistent with the idea that, in each of these cases,_. only a single contiguous portion of the Y chromosome is present. That is, the Y-specific sequences can be ordered so that, in each of the patients tested, the Y sequences present are a single, uninterrupted cluster. In each of the persons with cytogenetically detected abnormalities the chromosome banding studies are consistent with the presence of a single, contiguous portion of the Y chromosome. Such studies are not definitive and are not sufficiently precise to exclude more complicated rearrangements of the Y chromosome.

However, the patterns of Y-specific DNA sequences present in the individuals tested make it possible to generate a consistent deletion map of the human Y chromosome. In this map, each of the Y-specific restriction fragments tested is assigned to one of ten deletion intervals, as shown in Figure Figure 1a, as mentioned above, is a deletion map in which an inversion polymorphism is represented; the order of intervals 3B and 4A are reversed in this map.

Earlier evidence suggested that many human XX males contain Y-specific DNA sequences but are heterogeneous with respect to the amount of Y-chromosomal material present. Guellaen, G. et al., Nature, 307: 172-173 (1984); Page, D.C. et al., Nature, 315: 224-226 (1985). The present work extends that evidence: Y-specific DNA sequences were detected in the genomes of 12 of 19 XX males tested. In all cases, the Y-specific restriction fragments observed are of the same lengths as in normal males. The 12 Y(+) XX males can be divided into five classes according to the number of Y-specific fragments present. The DNA sequences present in the first four classes comprise a nested series (see Figure 1). The Class 1A XX male carries only one of the Y-specific restriction fragments for which testing was done (defining interval 1A in Figure 1). Class 1B XX males carry that same Y-specific fragment and an additional fragment (1B). Class 2 XX males carry these same Y-specific fragments, as well as thirteen others (defining interval 2). Class 3 XX males carry all the Y-specific fragments present in Class 2 as well as eight others (defining interval 3). It is also possible to orient the Y-chromosome deletion map with respect to the arms (Yp and Yq) and the centromere, as a result of hybridization data.

Five males with microscopically visible deletions of the long arm of the Y (and an apparently intact short arm) and one female with a translocation of Yq heterochromatin to chromosome 15 were among those scored for the presence or absence of Y-specific restriction fragments. The sterile 46,XYq- males lack the DYZ1 and DYZ2 repeated sequences as well as certain single-copy Y sequences. These sterile 46,XYq- males can be divided into two classes according to the number of single-copy Y sequences they lack. One case (CHM018) differs from the others (CHM004; SL; LL92) by the presence of the 8-kb Taql fragment detected by probe 12f (specific for interval 5 in Figure 1). A normal male with a non-fluorescent Y chromosome (CHM007) lacks only the DYZ1 and DYZ2 repeats (specific for interval 7) and differs from case CHM018 by the presence of several single-copy sequences (specific to interval 6). Conversely, a 46,XX,der (15), t(Y;15) (q12;pll) female, with the quinacrine-bright distal portion of Yq translocated to chromosome 15, carries the DYZ1 and DYZ2 repeats (interval 7) but none of the Y-specific single copy sequences listed in Table 1 (intervals 1 through 6). However, another Y-specific single copy sequence has been detected in this female.

These Yq deletions make it possible to assign intervals 5, 6, and 7 to the long arm of the Y chromosome (Figure 1). They thus serve to orient the deletion map with respect to the long and short arms of the Y chromosome. Assuming that these Yq-chromosomes represent simple deletions (and not, for instance, translocation products), they all have the Y centromere. On the other hand, it seems unlikely that the Y(+) XX males, in whom no Y chromosomal was detected cytogenically, have the Y centromere. The Y centromere is found, then, in interval 4B, which is present in all the XYq(-) males but absent in all the XX males (Figure 1). Interval 4 also contains the Y locus detected by probe pDP34 (DXYSl). This locus has been mapped to Yp (the short arm of the Y chromosome) by in situ hybridization. Page, D.C. et al., Nature, 311: 119-123 (1984). Interval 4 therefore appears to include part of the short arm as well as the centromere of the Y chromosome; interval 4A has been assigned to Yp. It follows that intervals 1 to 3 are entirely within Yp. These conclusions are based on the assumption that the rearrangement in the Yq- chromosomes is a simple deletion affecting Yq only. While the morphology of each Yq(-) chromosome is compatible with such a deletion, more complicated rearrangements cannot be excluded on the basis of cytogenetic studies. If events such as inversions or translocations were involved, the above conclusions might have been modified. The Y-specific DNA sequences which are present in Y(+) XX males are those in intervals 1 through 3B, which have been assigned to Yp. Class 4 Y(+) XX males also have sequences in interval 4A. All of the Y(+) XX males (and all of the XYq- males) have interval 1A in common. Assuming that XX males have testes because of the presence of a male-determining portion of the Y chromosome, that male determinant can be mapped to interval.1A. This assignment of the male determinant to Yp is in agreement with most karyotype-sex phenotype correlations. Davis, R.M., Journal of Medical Genetics, 18: 161-175 (1981).

The work described herein provides considerable information as to the organization of DNA sequences within the Y chromosome. Under the hybridization conditions used, probes 50f2, 52d, and 118 detect multiple Y-specific restriction fragments. The fragments detected by each of those probes are not all clustered within single intervals. In Class 3 XX males, bands 52d/C and 52d/B are of equal intensity; in normal males (or in the XYq-males), 52d/C is more intense than 52d/B. It is concluded that band 52d/C is at least a doublet, composed of one copy found in interval 3 and one or more copies in interval 4.

Probe 118 detects many Y-specific Taql fragments; of the four Y-specific fragments which could be scored unambiguously, three (118/A,B,C) map to interval 3, while one (118/D) maps to interval 6 Thus, probes 50f2 and 52d each detect sequences in intervals 3 (on Yp), 4, and 6 (on Yq); probe 118 detects sequences in intervals 3 and 6 (and probably elsewhere). These Yp-Yq homologies may be the result of one or more duplications of portions of the Y chromosome during evolution. Families of highly homologojιs DNA sequences have also been observed on the mouse Y chromosome. Lamar, E.E. and

E. Palmer, Cell, 37: 171-177 (1984); Bishop, CE. et al., Nature, 315: 70-72 (1985).

Example 2 Hybridization of Two DNA Probes

Detecting Yq(-) Specific Sequences to XX Male DNA and XYq(-) Male DNA

Two labeled probes were hybridized to a gel transfer of Taql-digested DNAs from a normal female, an XX male, two XYq- males, and a normal male. Figure 2 is an autoradiogram showing results of these hybridizations. The left panel of Figure 2 shows the results of hybri .di.zati.on of 32P-labeled probe 49f to the gel transfer. 49f detects two autosomal bands present in all of these individuals. 49f also detects a Y-specific 2.0-kb fragment in all normal males tested and in one of the two XYq- males shown in Table 1 above (case 26/CHM007). 49f detects several additional Y-specific fragments; the size and presence of some of these is polymorphic, as can be seen in the comparison of XYq(-) male 26 (CHM007) with a normal male (see Table 1). These and the other individuals listed in Table 1 were scored for the presence or absence of the nonpolymorphic Y-specific 2.0-kb fragment (indicated in the left panel by an arrow). Probe 12f was hybridized to the same Taql gel transfer. 12f detects many autosomal and X-chromosomal fragments present in all of these individuals. In addition, 12f detects several Y-specific fragments; individuals were scored for the presence or absence of the 8-kb fragment (indicated in the right panel of Figure 2 by an arrow) .

Example 3 Hybridization of Probes 47a and 47z to DNA of an XX Male Two ³²P-labeled probes, 47a and 47z, were hybridized to gel transfers of Taql-digested DNAs from a normal male, a normal female, and XX male 10 (CON 101). Figure 3 is an autoradiogram showing results of these hybridizations. Results of hybridization with 47a are shown in the left panel of Figure 3. 47a detects an X-chromosomal fragment of 8 kb and Y-specific fragment of 3 kb (shown by an arrow in Figure 3). Results of hybridization with 47z are shown in the right panel. 47z detects X-chromosomal fragments of 1.5 kb and 2.1 kb and a Y-specific fragments of 4,3 kb (shown by an arrow in Figure 3). Individuals were scored for the presence or absence of the 3 kb fragment and the 4.3 kb fragment; results are shown in Table 1 (above).

Example 4 Use of Y-specific DNA Hybridization

Probes to Explain Maleness in Two 45, X Males

As discussed above, very rare 45,X individuals are sterile males with testes. The genetic basis for their maleness cannot be determined using chromosome banding studies. In this example, the use of DNA hybridization probes to detect X-linked restriction fragment length polymorphisms (RFLP) and/or Y-specific restriction fragments in genomic DNAs from two 45,X males and their relatives is described. Subjects

Two 45, X males from two families and their mothers and fathers were tested, as was the brother of one of the 45, X males. Cytogenetic Studies Patient 1

Both parents were cytogenetically normal, as shown by lymphocyte and skin fibroblast mitotic studies; the father was shown to be 46,XY and the mother to be 46, XX. The proband was studied repeatedly. Four blood cultures and one fibroblast culture tested between 1971 and 1979 showed 45, X mitoses only. Several hundred mitoses and 1000 interphase nuclei from the 1979 blood culture were screened by quinacrine fluorescence for the presence of a Y chromosome or a Y chromatin body, but neither was found.

In 1971 a buccal mucosa smear had shown 15/1000 cells with a fluorescent spot judged at the time to be a possible Y chromosome. Again in 1977 some 5% of buccal mucosa cells appeared to be Y chromatinpositive.

Skin fibroblast studies in 1982 showed that though most mitoses (179/186) were 45, X, 5/186 cells were clearly 46, XY aas seen in G-banding by trypsin (GTG) and Q-banding by quinacrine fluorescence

(QFQ). QFQ banding indicated that the Y chromosome was structurally normal with a brightly fluorescent band Yq12 and that it was the same length (longer than average) as the father's. Moreover, 34/1000 of interphase nuclei from the same culture has a Y-chromatin body corresponding in size to the Y chromosome. In 1984 a frozen aliquot of the same fibroblast culture was thawed and the chromosome studies repeated. Of a total of 434 mitoses studied by quinacrine fluorescence, 5 had the karyotype 46,XY while all others had less than 46 chromosomes and no Y chromosome. Moreover, 8/1000 interphase nuclei had a Y chromatin body. The DNAs used for the detection of Y chromosome-specific DNA sequences were prepared from these 1982 and 1984 fibroblast cultures. Patient 2

Both parents and the brother were cytogenetically normal; the father and the brother were shown to be 46,XY and the mother to be 46, XX. Only 45,X cells were detected in the propositus, in blood cultures (on 2 occasions) and skin fibroblast cultures (on 3 occasions). In addition, 1000 interphase nuclei from the 1975 blood culture, 1000 nuclei from a buccal smear, and 100 mitoses and 1000 interphase nuclei from the blood culture made in 1979 were screened by quinacrine fluorescence for the presence of Y chromatin or a Y chromosome, but none was found. Thus, there was no indication of the presence of any Y chromosome in this patient. DNA Studies Human genomic DNAs, prepared from peripheral leukocytes or cultured skin fibroblasts, were analyzed by restriction digestion, agarose electrophoresis, gel transfer, and hybridization with radiolabeled cloned DNA probes. The DNA hybridization probes used detect X-linked restriction fragment length polymorphisms (RFLP) and/or Y-specific restriction fragments.

To determine the parental origin of the single X chromosome in the two 45, X males, both families were typed for as many as eight X-linked RFLPs. These X-linked RFLPs provide information as to the parental origin of the X chromosome only when, by chance, the propositus is hemizygous for an allele present in only one parent. The probes used for this purpose are as follows:

1. RC8 detects X-linked, allelic Taq I fragments of 3.0, 3.4 and 5.7kb.

2. D2 detects X-linked, allelic Pvu II fragments of 6.0 and 6.6kb.

3. L1.28 detects X-linked, allelic Taq I fragments of 10 and 12kb. 4. pDP34 detects X-linked, allelic Taq I fragments of 11 and 12kb as well as a Y-specific 15kb fragment. 5. 19-2 detects X-linked, allelic Msp I fragments of 4.4 and 12kb. 6. S21 detects X-linked, allelic Taq I fragments of 2.5 and 2.7kb.

7. 22-33 detects X-linked, allelic Taq I fragments of 10 and 17kb.

8. 43-±5 detects X-linked, allelic Bgl II fragments of 6 and 9kb.

The results are shown in Table 2 (below).

In both cases, the propositus' single X chromosome was demonstrated to be maternal in origin.

Hybridization probes derived from the human Y chromosome were used to test the two 45, X males and their relatives for the presence of a number of Y-specific sequences. The probes used for this purpose are as follows : 9. 47c detects Y-specific Taq I fragments of 3 and

4.3kb. 10. 115 detects a Y-specific Taq I fragment of either 2.1 or 2.6kb (D.C. Page and J. Weissenbach, unpublished results). 11. 50f2 detects multiple Y-specific loci on Eco RI or Taq I digests. 12. 52d detects multiple Y-specific loci on Eco RI or Taq I digests. 13. A 1.8-kb PstI fragment purified from plasmid

71-7A detects multiple Y-specific Taq I fragments. 14. pY431-HinfA detects a highly repeated Y- specific Hae III fragment of 2.1kb. 15. pY3.4 detects a highly repeated Y-specific Hae

III fragment of 3.4kb. Probes 47c, 115, 50f2, 52dpDP34 and 71-7A detect single-copy, Y-specific sequences. Three of them (50f2, 52d and 71-7A) detect multiple Y-specific restriction fragments. Probes pY431-HinfA and pY3.4 detect Y-specific repeated sequences. The results are shown in Table 3 (below). In both families, the normal 46, XY males (the fathers and the brother) exhibit all of the Y-specific sequences found in unrelated control males. The mothers (normal 46, XX karyotypes) exhibit none of these Y-specific sequences.

None of the single-copy, Y-specific sequences tested for were detected in either of the 45,X males. In patient 1, the highly repeated, Y-specific 2. Ikb and 3.4kb Haelll fragments homologous to probes pY431-HinfA and pY3.4 were detected.

However, these Y-specific repeated sequences were present in greatly reduced amounts, in comparison with amounts detected in the father of patient l or in unrelated control males. To quantitate the reduction of these repeated sequences, the intensity of the hybridizing 2.1- and 3.4kb Hae III fragments in patient 1 was compared with the corresponding intensities obtaining using equal or reduced (10-fold, 100-fold, 1000-fold, and 10, 000-fold) amounts of paternal DNA. The intensity of the 2.1-kb Hae III band in the 45,X male is somewhat greater than that observed with paternal DNA in 100-fold reduced amount (Figure 2). A similar reduction in the intensity of the 3.4kb Hae III band in this 45, X male was also observed. Both Y-specific 2. Ikb and 3.4kb Hae III fragments are present in about 1 to 3% of the amount present in the father. No trace of these Y-specific repeated sequences was found in patient 2, even when conditions were used in which it is possible to detect the presence of normal male DNA in 10,000-fold reduced amount (i.e., the presence of a normal Y chromosome in as few as 1 in 10 , 000 cells . ) .

However, these repeated Hae III fragments are located principally if not exclusively in distal Yq, and thus would be of little use in detecting mosaicism involving an abnormal Y chromosome lacking that region. The DNA hybridization studies alone, then, cannot argue against low-grade mosaicism for a structurally abnormal Y chromosome in patient 2. Similarly, cytogenetic methods based on detection of the quinacrine-bright distal portion of Yq cannot argue against such mosaicism.

Example 5 Detection of Small Deletions of the

Short Arm of the Y Chromosome in 46, XY Females

Subjects

Case 1 has Turner stigmata. Her gonads, which were removed at age 4, were streaks which consisted of dense ovarian stroma with no primordial follicles or testicular tissue. A skin fibroblast culture established from this patient was found to be borderline positive for H-Y antigen after a repeat test using two different assays.

Case 2 has several features of Turner syndrome. She has congenital lymphedema. She had primary amenorrhea and developed bilateral gonadoblastoma. Histological examination of the gonads showed gonadoblastoma and streaks with no primordial follicles. A gonadal fibroblast culture established from this patient was H-Y antigen positive. Cytogenetic Studies

Cytogenetic analysis was performed on peripheral blood samples from cases 1 and 2 and on fibroblast cultures from a small skin biopsy of case 1 and from a gonadal biopsy of case 2. Prometaphase cells were stained by G-banding, R-banding, C-banding and Q-banding. A small deletion of the short arm of the Y chromosome [46,X, del (Y) (pll)] was identified in both patients. The deletions were barely detectable on metaphase chromosomes. It was not possible to determine whether the deletions were interstitial or terminal, or whether they differed in the two patients. The long arm of the Y chromosome of both patients appeared of normal size, including a Q-bright heterochromatic region of average size. The Y chromosome of the father of each patient was normal. No other karyotypic abnormalities were identified in either patient and no evidence of mosaicism was found in the tissues analyzed: in case 1, 105 cells and 101 cells were examined in the blood and skin samples, respectively, while in case 2, 120 cells were examined in the blood and 54 and 58 cells in bilateral gonadal samples, respectively. DNA Studies

DNA was prepared from blood samples and fibroblast cultures of both patients, the father of case 2 and normal control male and female individuals. The DNAs were digested to completion with the restriction endonucleases Taql (for probe pDP34) or EcoRI (for probes 50f2, 52d, 47b, 118, p12f2 and p12f3). DNA fragments separated on agarose gel electrophoresis were transferred to membrane filters for hybridization by Southern blot technique. Seven

³² P-labeled DNA probes that have been mapped to the

Y chromosome were hybridized to patient and control genomic DNA blots. (See I above and Example 1)

Probe pDP34 (DXYS1) detects homologous sequences on the short arm of the Y chromosome and on the long arm of the X chromosome. Probes 50f2 and 52d hybridize with DNA sequences both on the Y chromosome and on autosomes on the X chromosome. Probe 47b detects Y-, X- and autosomal sequences; probe 118 is exclusively Y-specific. Two additional probes, p12f2 and p12f3 are located on the long arm of the Y chromosome. Results of these analyses can be summarized as follows:

1. Hybridization of probe pDP34 showed that patient 1 had a hybridization pattern characteristic of a normal male (DXYS1 was not deleted) and patient 2 was missing the 15kb Y-specific bond (i.e., Y-specific DNA homolgous to DXYSl was deleted).

2. Hybridization of probes 118, 52d, 50f2 and 47b showed that patient 1 was missing six of the male-specific bands with all four probes (confirming cytogenetic data indicating a deletion of a portion of the

Y chromosome) and patient 2 had a missing

Y specific band with probe 50f2 and probe 47b (the latter identical to that missing in patient 1).

3. Hybridization with probes p12f2 and p12f3 (located on the long arm of the Y chromosome) showed normal male hybridization patterns for both patients. Results of the hybridization of probe pDP34 to patient and control DNAs were compared. Case 1 showed a hybridization pattern characteristic of a normal male with bands at 11 and 15 kb, indicating that DXYSl was not deleted in this case. Case 2, however, was missing the band at 15 kb, indicating that the Y-specific DNA homologous to DXYSl had been deleted in that patient. The father of the latter patient showed normal Y- and X-specific bands at 15 and 12 kb, respectively.

The hybridization of ³²P-labeled probes 118, 52d, 50f2 and 47b to EcoRI digested DNA's from normal female, normal male, case 1, the father of case 2 and case 2 was also assessed. Case 1 showed deletions of a total of six of the male-specific bands with all four probes 118, 52d, 50f2 and 47b. This confirmed the cytogenetic data which indicated that case 1 had a deletion of a portion of the Y chromosome. Probes 118, 52d and 50f2 showed some male-specific bands that were not deleted in case 1 as compared to a normal male. This indicates that these three probes presumably recognized sequences located in other regions of the Y chromosome than that deleted in case 1.

Case 2 did not appear to be missing Y-specific bands with probes 118 and 52d. With probe 50f2, both the father of case 2 and case 2 had a missing

Y-specific band (indicated with a small arrow) which may represent a polymorphic band. With probe 47b, case 2 showed a missing Y-specific band identical to the one missing in case 1 but present in normal males, including the father of case 2. Studies with two additional probes, p12f2 and p12f3, which are located on the long arm of the Y chromosome showed normal male hybridization patterns for both patients 1 and 2.

Table 4 summarizes the hybridization data which indicate that cases 1 and 2 have different deletions. The only band these patients are missing in common is homolgous to probe 47b. This probe is adjacent to probe 47c, which was most often present in patients who are 46, XX males. One or both of the deletions described here are likely to be interstitial, extending on either side of the location of 47b.

The two patients have different but overlapping deletions which could explain that while both patients have features of Turner syndrome, they differ in some respects. Case 1 has short stature, while case 2 is of normal height: this may indicate the presence of genes controlling height on the short arm of the Y chromosome, which are deleted in case 1 but not in case 2. In addition, case 2 but not case 1 developed gonadoblastoma; however the gonads were removed at an early age in case 1. HY antigen was definitely present in only case 2, indicating that the absence of different regions of the short arm of the Y chromosome affects HY antigen expression, independently of male diffentiation. Independence of HY antigen and male sexual differentiation was recently described in mouse. HY antigen was not consistently positive or negative in cases of 46,XY pure gonadal dysgenesis, a condition likely to have several causes. Page, D.C. et al., Nature: 311; 119-123 (1984). Thus, use of the DNA probes described, which are homologous to regions on the short and the long arms of the Y chromosome, demonstrated that the sex chromosomal anomaly, the 46,XY female, resulted from small deletions of the short arm of the Y chromosome. That is, small deletions of the short arm of the Y chromosome were detected; no deletions on the long arm were detected with the probes used. The deletions, although different, included a common overlapping region (detected by probe 47b) apparently essential for male differentiation. In addition, HY antigen was definitely present only in patient 2, indicating that the absence of different regions of the short arm of the Y chromosome affects HY antigen expression, independently of male differentiation. The results are summarized below in Table 4.

Industrial Applicability

DNA probes which detect DNA sequences homologous to those occurring in the normal Y chromosome and methods of using them in assessing the presence or absence of Y DNA in individuals can be used in a clinical or other medical context for diagnosis and evaluation of many postulated functions. For example, they can be used for the assessment of suspected chromosomal abnormalities, the determination of the genetic basis of phenotypic abnormalities and the diagnosis of genetic disorders and their related effects.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. Isolated DNA wh i c h hybridizes to at least a portion of one of the Y chromosomal intervals of Figure 1.

2. Isolated DNA of Claim 1 which hybridizes to the Y chromosomal interval in which the testis determining factor is located.

3. Isolated DNA of Claim 2 which has a detectable label.

4. Isolated DNA sequences which hybridize to the short arm of the normal human Y chromosome.

5. Isolated DNA sequences which hybridize to the long arm of the normal human Y chromosome.

6. Isolated DNA which hybridizes to the region of the normal human Y chromosome required for spermatogenesis.

7. Isolated DNA of Claim 6 which hybridizes to the long arm of the normal human Y chromosome.

8. Isolated DNA sequences which are Y-linked restriction fragment length polymorphisms.

9. Isolated DNA sequences which detect pseudoautosomal loci common to the X chromosome and the Y chromosome.

10. In a method of detecting Y-specific DNA in human sex chromosomes, the improvement comprising the use of DNA probes which hybridize to sequences homologous to sequences present in the normal Y chromosome.

11. DNA probes for the detection of human sex chromosomal anomalies, the probes comprising DNA which hybridizes to DNA sequences homologous to the sequences of the normal Y chromosome.

12. DNA probes of Claim 11 comprising at least one probe for each of the intervals in the deletion map of the normal Y chromosome represented in Figure 1.

13. A probe which detects the presence or absence of DNA sequences homologous to DNA sequences of the normal sex chromosome, said probe comprising a vector having therein a restriction fragment of the normal sex chromosome or equivalent sequences.

14. A probe of Claim 12 comprising a vector which is a plasmid having inserted at its Hindlll site a restriction fragment which is an 0.9Kb Hindlll fragment of the Y chromosome or equi-valent sequences.

15. A probe of Claim 12 comprising a vector which is a plasmid having inserted at its Hindlll site a restriction fragment which is a 3 .5Kb Hindlll fragment of the Y chromosome or equivalent sequences.

16. A probe of Claim 12 comprising a vector which is a plasmid having inserted at its AccI and

EcoRI sites a restriction fragment which is a 1.0Kb EcoRI/TaqI fragment of the Y chromosome or equivalent sequences.

17. A probe of Claim 12 comprising a vector which is a plasmid having inserted at its Hindlll site a restriction fragment which is a 4.6Kb Hindlll restriction fragment of the Y chromosome or equivalent sequences.

18. A method of detecting the presence of Y- specific DNA in a sample, comprising the steps of: a. preparing the sample so as to render DNA present in the sample available for hybridization; b. contacting the DNA in the sample with DNA sequences which hybridize with Y chromosome DNA under conditions suitable for hybridization to occur; and σ. determining the hybridization of the DNA in the sample with the DNA sequences which hybridize with Y chromosome DNA.

19. A method of Claim 18 in which the DNA sequences which hybridize with Y chromosome DNA have a detectable label.

20. A method of Claim 19 in which the DNA sequences which hybridize with Y chromosome DNA is radioactively or chemically labelled.

21. A method of detecting in a sample the presence of DNA sequences homologous to those in the normal Y chromosome, comprising the steps of: a. rendering DNA in the sample available for hybridization; b. contacting the DNA in (a) with DNA probes which hybridize with at least one interval in the deletion map of the normal human Y chromosome, under conditions appropriate for hybridization of DNA in the sample with the DNA probes; and c. detecting the hybridization of DNA in the sample with the DNA probes.

22. A method of Claim 21 in which the DNA probes are labelled.

23. A method of Claim 22 in which the DNA probes are radioactively, enzymatically or chemically labelled.

24. A method of detecting the occurrence of normal human Y chromosome DNA in an individual, comprising the steps of: a. contacting DNA from the individual with a set of DNA hybridization probes under conditions suitable for hybridization to occur, the set being comprised of DNA probes for each of the intervals in the deletion map of the normal human Y chromosome of Figure 1 and the probes comprising DNA which hybridizes to DNA sequences in the normal human Y chromosome; and b. detecting hybridization of DNA from the individual with the DNA probes.

25. A method of Claim 24 in which the DNA probes are selected from the group consisting of: pDP307, pDP132, pDP61, pDP125, 50f2, pDP34, 12f and pY431-HinfA.

26. A method of detecting the presence in human sex chromosomes of DNA sequences encoding the testis-determining factor, comprising the steps of: a. contacting DNA from human sex chromosomes with DNA hybridization probes which are DNA sequences encoding the testis-determining factor, under conditions suitable for hybridization to occur; and b. detecting hybridization of the DNA from sex chromosomes with the DNA hybridization probes.

27. In a method of assessing sex chromosomal anomalies, the improvement comprising the use of DNA hybridization probes which DNA sequences of the normal human Y chromosome and hybridize to at least a portion ofone of the Y chromosomal intervals of Figure 1.

28. A DNA hybridization probe for detection of the testis determining factor in human Y chromosomal DNA, the probe comprising an 0.9Kb Hindlll fragment from normal Y chromosome DNA, subcloned into the Hindlll site of pUC-13.