Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Incontinentia pigmenti (type 1) and X;5 translocation.

The authors present a 5-year-old girl with total absence of speech, dysmorphic features, pigmented lesions on the legs, an abnormal EEG and otherwise normal intelligence representing a mild form of type 1 Incontinentia pigmenti associated with an (X;5) (p11.2;q35.2) apparently balanced translocation prenatally diagnosed. The seven previous translocation type 1 IP patients are reviewed and all have the same Xp11 breakpoint. Somatic cell hybrids have been made to further study this breakpoint and further define the putative type 1 IP gene.     

Physical mapping at a potential X-linked retinitis pigmentosa locus (RP3) by pulsed-field gel electrophoresis.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common SfiI fragment of 205 kb. As the CGD gene covers approximately 30-60 kb, the RP3 locus has been restricted to approximately 150-170 kb. A CpG island, potentially marking a new gene, was identified within the SfiI fragment at a position approximately 35 kb from the deletion endpoint in BB.     

Isolation of DNA markers from a region between incontinentia pigmenti 1 (IP1) X-chromosomal translocation breakpoints by a comparative PCR analysis of a radiation hybrid subclone mapping panel.

A strategy based on the use of human-specific interspersed repetitive sequence (IRS)-PCR amplification was used to isolate regional DNA markers in the vicinity of the incontinentia pigmenti 1 (IP1) locus. A radiation hybrid (RH) resulting from a fusion of an irradiated X-only somatic cell hybrid (C12D) and a thymidine kinase deficient (TK-) hamster cell line (a23) was identified as containing multiple X chromosome fragments, including DNA markers spanning IP1 X-chromosomal translocation breakpoints within region Xp11.21. From this RH, a panel of subclones was constructed and analyzed by IRS-PCR amplification to (a) identify subclones containing a reduced number of X chromosome fragments spanning the IP1 breakpoints and (b) construct a mapping panel to assist in identifying regional DNA markers in the vicinity of the IP1 locus. By using this strategy, we have isolated three different IRS-PCR amplification products that map to a region between IP1 X chromosome translocation breakpoints. A total of nine DNA sequences have now been mapped to this region; using these DNA markers for PFGE analyses, we obtained a probe order DXS14-DXS422-MTHFDL1-DXS705. These DNA markers provide a starting point for identifying overlapping genomic sequences spanning the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should assist the molecular analysis of this locus.     

A recombination outside the BB deletion refines the location of the X linked retinitis pigmentosa locus RP3.

Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was thought to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, and the McLeod phenotype. This patient carried a deletion extending approximately 3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located approximately 40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected mate shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3.     

Two cases of X/autosome translocation in females with incontinentia pigmenti

Summary We report two unrelated girls who present some clinical features of severe incontinentia pigmenti (IP), with characteristic skin pigmentation. Both have balanced de novo X/autosome translocations involving band Xp11. The coincidence of the probable de novo expression of an X-linked disorder in these two girls with translocations involving similar breakpoints on the X chromosome suggests that this band may be the site of the IP gene locus.     

Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system

The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such ”Pushmi-Pullyu” hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from ∼2 Mb to ∼20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with ∼2–3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with ∼14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres. Received: 15 December 1998; in revised form: 2 March 1999 / Accepted: 5 April 1999     

A somatic cell hybrid panel to facilitate identification of DNA sequences in the vicinity of the incontinentia pigmenti locus (IP1)

Somatic cell hybrids that retain derivative X chromosomes from women with sporadic incontinentia pigmenti (IP1) and de novo X/autosomal translocations with consistent breakpoints at Xp11.21 were constructed. An assembled hybrid panel was used to physically map DNA sequences in relationship to the IP breakpoint. DSX14 was found to map to region Xp11.21----p11.1. Regional assignments of 19 X-chromosomal loci were reviewed.     

Molecular cloning of Xp11 breakpoints in two unrelated mentally retarded females with X;autosome translocations

Mental retardation is a very common and extremely heterogeneous disorder that affects about 3% of the human population. Its molecular basis is largely unknown, but many loci have been mapped to the X chromosome. We report on two mentally retarded females with X;autosome translocations and breakpoints in Xp11, viz., t(X;17)(p11;p13) and t(X;20)(p11;q13). (Fiber-) FISH analysis assigned the breakpoints to different subbands, Xp11.4 and Xp11.23, separated by approximately 8 Mb. High-resolution mapping of the X- chromosome breakpoints using Southern blot hybridization resulted in the isolation of breakpoint-spanning genomic subclones of 3 kb and 0. 5 kb. The Xp11.4 breakpoint is contained within a single copy sequence, whereas the Xp11.23 breakpoint sequence resembles an L1 repetitive element. Several expressed sequences map close to the breakpoints, but none was found to be inactivated. Therefore, mechanisms other than disruption of X-chromosome genes likely cause the phenotypes.     

Three Finnish incontinentia pigmenti (IP) families with recombinations with the IP loci at Xq28 and Xp11

The locus (IP2) for the hereditary form of incontinentia pigmenti (IP) has been mapped to Xq28 by linkage analysis. We studied three IP families with polymorphic markers in the Xq28 region. In two families we observed recombination between the marker loci and IP. In the third family no crossing overs were seen and linkage to the Xq28 region could not be excluded. The other IP locus (IP1) has been mapped to Xp 11.21, because of sporadic cases of IP with X-chromosomal alterations involving Xp11.21. To check whether this locus is linked to IP in these families, we used polymorphic markers in the Xp11 region. In all three families recombinations were observed, thus excluding linkage to this locus in these IP families.     

Genetic counselling in carriers of reciprocal chromosomal translocations involving short arm of chromosome X

A central concept in genetic counselling is the estimation of the probability of occurrence of unbalanced progeny at birth and other unfavourable outcomes of pregnancy (miscarriages, stillbirths and early death). The estimation of the occurrence probability for individual carriers of four different X-autosome translocations with breakpoints at Xp, namely t(X;5)(p22.2;q32), t(X;6)(p11.2;q21), t(X;7)(p22.2;p11.1), and t(X;22)(p22.1;p11.1), is presented. The breakpoint positions of chromosomal translocations were interpreted using GTG, RBG and FISH-wcp. Most of these translocations were detected in women with normal phenotype, karyotyped because of repeated miscarriages and/or malformed progeny. A girl with very rare pure trisomy Xp22.1-->pter and a functional Xp disomy was ascertained in one family and her clinical picture has been described in details. It has been suggested that not fully skewed X chromosome inactivation of X-autosome translocation with breakpoint positions at Xp22 (critical segment) could influence the phenotype and risk value. Therefore, the X inactivation status was additionally evaluated by analysis of replication banding patterns using RBG technique after incorporation of BrdU. In two carriers of translocations: t(X;5)(p22.2;q32) and t(X;7)(p22.2;p11.1), late replication state of der(X) was observed in 5/100 and 10/180 analysed cells, respectively. In these both cases the breakpoint positions were clustered at the critical segment Xp22.2. In two other cases, one with the breakpoint position within [t(X;22)(p22.1;p11.1)] and one outside the critical region [t(X;6)(p11.2;q21)], fully skewed inactivation was seen. Therefore, we suggest that neither the distribution of the breakpoint positions nor fully skewed inactivation influenced the phenotype of observed t(X;A) carriers. The occurrence probabilities of the unbalanced progeny were calculated according to Stene and Stengel-Rutkowski along with application of updated available empirical data. In the studied group the values of occurrence probability for unbalanced offspring at birth ranged from 2.1% to 17%. Information on the magnitude of the individual figures may be important for women carrying a reciprocal X;A translocation when deciding upon further family planning.     

Translocation (X;9)(p11;q34) in a girl with incontinentia pigmenti (IP): implications for the regional assignment of the IP locus to Xp11?

A t(X;9)(p11;q34) is reported in a girl with incontinentia pigmenti (IP). The X breakpoint is at p11.21. Although no similar case has been reported, this breakpoint may be significant insofar IP is considered an X-linked dominant mutation and could be of help in a specific X DNA probes study.     

Linkage relationships between X-linked retinitis pigmentosa and nine short-arm markers: exclusion of the disease locus from Xp21 and localization to between DXS7 and DXS14.

Linkage data between X-linked retinitis pigmentosa (XLRP) and nine X-chromosomal markers are reported. To test the assignment of XLRP to the Xp21 region (as considered at Human Gene Mapping 8), an analysis of XLRP and six markers flanking this region was undertaken. The XLRP locus was found to be excluded from the chromosome distal to ornithine transcarbamylase (OTC) (P = 6.5 X 10(-5]. Further data were accumulated with three more probes proximal to DXS7 (L1.28), the closest linked probe. Multipoint analysis of these data suggests a posterior probability of .94 that XLRP is proximal to DXS7 (L1.28), which has been mapped to the region Xp11.3.     

Array-CGH characterization of a de novo t(X;Y)(p22;q11) in a female with short stature and mental retardation

We report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facial dysmorphisms in combination with short stature. The structure of the derivative X chromosome was studied using BAC array-CGH which disclosed the Xp22 breakpoint between the STS and the VCX3A gene and the presence of the Yq11.1qter chromosome. It is common that females with Xp;Yq translocations present only short stature and are normal in every other aspect. Thus, this would be the first case in which a girl with Xp;Yq translocation presents an unusual phenotype with intermediate male clinical features with Xp;Yq translocations. The risk of developing gonadoblastoma in females with Y chromosome material is also discussed and, to this effect, different explanations related to this apparent variation are also presented.     

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11

Summary Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster x human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter-(DXS7, DXS146, DXS255)-IP1-(DXS14, DXS90)-DXS106-qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.     

Deletions in patients with classical choroideremia vary in size from 45 kb to several megabases

Making use of the p1bD5 probe (DXS165), we have isolated several markers from the choroideremia locus by chromosomal jumping, preparative field-inversion gel electrophoresis, and cloning of a deletion junction fragment. With these clones we were able to identify and characterize eight deletions in 69 choroideremia patients investigated. The deletions are heterogeneous, in both size and location. The smallest deletion (patient LGL1134) comprises approximately 45 kb of DNA, whereas the largest ones (patients 25.6 and LGL2905) span a DNA segment of at least 5 megabases, which is comparable in size to the smallest deletion detected in a TCD patient (patient XL45) showing a complex phenotype. The TCD deletions encompass variable parts of 150-200-kb DNA segment that is flanked by p1bD5 (DXS165) at the centromeric side and by pZ 11 at the telomeric side. The deletions in patients 33.1, LGL1101, and LGl1134 do not span a translocation breakpoint which was previously mapped on the X chromosome of a female with TCD. The clones isolated from the TCD locus are valuable diagnostic markers for deletion analysis of patients or carrier females. In addition, they should be useful for the isolation of expressed sequences that are part of the TCD gene.     

An 18-locus linkage map of the pericentromeric region of the human X chromosome: Genetic framework for mapping X-linked disorders

We report a high-resolution genetic linkage map of the region Xp11.4 to Xq13.3, spanning the centromere of the X chromosome and encompassing approximately 30 cM. This 18-locus map is composed of 11 intervals that are spaced on average about 3 cM apart. Markers incorporated into the map together detect 19 distinct polymorphisms and include five genes (TIMP, SYP, AR, CCG1, PGK1), the OATL1 cluster, the hypervariable locus DXS255, the centromeric locus DXZ1, and 10 other anonymous DNA segments. Given that this map spans roughly one-fifth of the length of the X chromosome and includes many loci currently used in both diagnosis and mapping of X-linked disorders, it should be useful for genetic counseling and for guiding efforts to clone disease genes in this region.     

Familial deletion of Xp21.2 with glycerol kinase deficiency and congenital adrenal hypoplasia

Summary Congenital adrenal hypoplasia (CAH) and glycerol kinase deficiency (GKD) were diagnosed in a male during the neonatal period. On prometaphase chromosomes there was an interstitial deletion involving Xp21.2 and possibly Xp21.3 in the propositus and his mother. Duchenne muscular dystrophy (DMD) was excluded on the basis of normal serum creatine kinase and a muscle biopsy. Molecular hybridization of DNA from the propositus with 11 probes covering Xp21, including the DMD locus, was normal. In situ hybridization with the probe pERT87.15 showed a normal signal at the expected site indicating that the DMD locus was preserved and not translocated. This suggests that the DMD locus is located at the most proximal part of the sub-band Xp21.2 or in Xp21.1, and that the DXS68 (probe L1) is far from it on the distal flanking DNA.     

Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis

Genetic and molecular studies show that the Duchenne muscular dystrophy (DMD) locus at Xp21 is large and complex. We have analyzed this region using pulsed field gel electrophoresis (PFGE) and have determined physical distances between Xp21 probes. The sum of the sizes of the Sfil restriction fragments detected by these probes is greater than 4000 kb. The deletion endpoints in two DMD patients were detected by observing changes in these restriction fragments. In addition, the Xp21 breakpoint for the X;1 translocation in an affected female was mapped. These results demonstrate the applicability of PFGE for analysis of Xp21, and should facilitate the mapping of other translocations and deletions in this region, some of which lead to glycerol kinase deficiency and adrenal hypoplasia as well as DMD.