The proband showed a 49,XXXXX karyotype in all cells without evidence of mosaicism (Figure 2). 5-bromodeoxyuridine (BrdU)-pulsed cultures showed clear patterns of asynchronic replication of the proband's X chromosomes (Figure 3); in all cells one early replicating X chromosome was identified, together with four late replicating X chromosomes showing different replication patterns. Replication patterns were classified as "early replicating" (e), "late replicating" (l) and "very late replicating" (vl) according to Sarto 35 . The observed proportion of cells showing different number of X replication patterns accounted for 31% with 1e/2l/2vl, 23% with 1e/3l/1vl, 17% with 1e/1l/3vl, and 29% with 1e/4vl.

Molecular analysis of five X-linked polymorphic loci in the proband and her mother (Table 1) allowed for the unequivocal identification of the parental origin of the X chromosomes in the proband who carried four maternal X chromosomes and one paternal X chromosome; this last one with a 173-108-199-206-141 (P) haplotype.

The methylation status of X chromosomes, assayed in the HUMANDREC region of the human androgen receptor gene (AR), showed a skewed pattern of inactivation in the proband's mother because one AR allelic fragment of 173 bp was preferentially amplified with respect to the other of 179 bp (Figure 4). Similar analysis in the proband showed that two maternal AR alleles were amplified (173 and 179); the 173 allele showing a preferential amplification with respect to allele 179, and lack of amplification of the paternal 199 allele (Figure 5). These results indicated (i) a preferential inactivation of maternal X chromosomes containing the 173 allele in the proband; (ii) the possibility that her maternal chromosomes with the 179 allele might be incompletely inactivated, and (iii) an active paternal X chromosome.

The segregation of the proband's X chromosomes in a panel of 51 HPRT1⁺ cell hybrid lines was informative for analysing the functional status of each individual X chromosome. Cell hybrids cloned in the same Petri dish were included only when they showed different molecular patterns; this was considered evidence that clones were different from one another. Conversely, when cell hybrids cloned in the same Petri dish showed an identical molecular pattern only one of them was included because we could not prove that they were different from one another. Table 2 lists the molecular patterns of 33 cell hybrid lines. Human-specific amplification patterns were not observed with rodent DNA with any primer herein tested under similar PCR conditions.

Analysis of cell hybrids allowed for the identification of three maternal haplotypes: M1, with 185-112-173-202-135 (in cell hybrids 1PXKB2, 1PXKB5, 1PXKF4, 1PXKH4, 1PXKI3, 1PXKL1, 1PXKN2); M2, with 163-104-179-204-139 (in cell hybrids 1PXKG4, 1PXKH2, 1PXKJ6, 1PXKM1 and 1PXKR1), and MR, with 185-112-179-204-139 (in cell hybrid 1PXKQ3). MR was a recombinant maternal haplotype, derived from a crossover between DXS1068 and AR. The finding of cell hybrid lines with single M1, M2 or P haplotypes, indicated that the HPRT1 locus of donor origin was functionally active in different X chromosomes of the proband.

To discriminate whether survival in HAT medium was associated to presence of one (or more) active X chromosome(s) in cell hybrids showing any combination of two or three haplotypes, DNA from cell hybrids was first digested with methylation-sensitive enzymes and subsequently amplified with AR-C primers (see Table 3).

These results showed that in nine cell hybrids with M1-M2 haplotypes, the 173 allele was exclusively amplified in seven cell lines. In one cell hybrid line (1PXKA1), both maternal AR alleles (173 and 179) were amplified, showing similar peaks and suggesting random inactivation, while in another cell hybrid line (1PXKP3), the 179 allele was exclusively amplified. In all five cell hybrids with the M1-P haplotypes, the 173 maternal allele was exclusively amplified, as was the 179 maternal allele in the two M2-P cell hybrid lines. Similarly, the 179 maternal allele was exclusively amplified in 1PXKQ3, a cell hybrid with a recombinant maternal haplotype (MR) and a paternal (P) haplotype. In ten other cell hybrid lines with the M1-M2-P haplotypes, the observed pattern of amplification was similar to the one observed in the proband's lymphocytes, showing a large peak corresponding to allele 173, a smaller peak corresponding to allele 179, and lack of amplification of the 199 paternal allele.

A comparison with FISH data showed that the number of human X chromosomes retained in cell hybrids and the number of X haplotypes were very frequently coincident in a sample of eight cell hybrid lines in which all possible haplotype combinations were observed Table 3; Figure 6). This correspondence was also found in cell hybrid 1PXKJ6, in which a single haplotype and a single human X chromosome were observed.

Sequence analysis of the minimal region of the XIST promoter did not show C(- 43)A or C(- 43)G in the proband and her mother, ruling out that alterations at this site might be responsible for skewed X inactivation. Moreover, mutational screening of the proband's X chromosomes in 10 cell hybrids confirmed that these mutations were not present in her chromosomes. This was especially evident in three of these cell hybrids (1PXKB5, 1PXKE3 and 1PXKG4), each which with a single X haplotype (M1, P and M2 respectively).

C.S.S is a 15 year old female whose mother was 17 years of age at the time she was born. At birth, she weighted 1,200 g and her height was 39 cm. She was referred for clinical investigation for multiple anomalies (congenital heart defect, cleft soft palate, and facial dysmorphies) at 4 months of age. A recent physical examination showed a mentally retarded young adult, with poor language development, marfanoid habitus and disproportionate upper/lower body segments (Figure 1); her profile was very flat; with ocular hypertelorism, upslanting palpebral fissures, epicanthic folds, flat nasal bridge, low-set ears, posterior cleft palate, retrognathism, and micrognathia. A low posterior hairline, left scoliosis, bilateral fifth finger clinodactily, hand and feet arachnodactily, and difficulty on supination of the right arm corresponding to a radioulnar synostosis were present. A congenital heart defect (patent ductus arteriosus and ventricular septal defect) was surgically repaired at the first year of life. A low count of dermal ridges was also present.

Cytogenetic analyses were carried out at 4 months and at 15 years of age using standard lymphocyte cultures and BrdU-pulsed cultures 43 . Chromosome preparations were identified by conventional GTG banding 44 at the 500 band level in 100 cells. Chromosome preparations of BrdU-pulsed cultures were stained 45 , and X replication patterns identified in 100 cells and classified according to Sarto's criteria 35 .

Hprt - cell lines of rodent origin were used as recipient cell lines in fusion experiments with human lymphocytes of the proband isolated with Ficoll^® (Sigma, St Louis). A cell hybrid panel was prepared with AKO1/15 cells derived from Akodon cursor 46 . Following a seven day growth in DMEM/6MP selective medium (Dulbecco's Minimal Essential Medium supplemented with 10% FCS and 6-mercaptopurine 16.7 μg/ml), 3 × 10⁶ recipient cells were fused with 1.2 × 10⁷ human lymphocytes 47 . Cell hybrids were selected in DMEM/HAT medium [DMEM with 10% FCS, hypoxanthine (100 μM), aminopterin (0.4 μM), thymidine (16 μM)].

Cell death was monitored periodically; after a 2-3 weeks period, visible colonies were cloned inside Penicillinders, transferred to individual dishes and expanded to culture flasks. A maximum of three colonies was cloned from each Petri dish. Each dish was identified by a letter and each colony by a number. Controls were carried out for demonstrating adverse selection of recipient cells; aliquots of the same AKO1/15 cells used in fusion experiments were grown in DMEM/HAT medium.

DNA was extracted from blood samples of the proband, her mother, the recipient cell line AKO1/15, and cell hybrid lines. DNA isolation was carried out by standard procedures 48 .

PCR amplifications of four human, X-linked microsatellite loci were carried out with DNA samples of the proband, her mother, 51 cell hybrid lines, and the rodent cell line. PCR reactions contained 100 ng of DNA, reaction buffer [16 mM (NH₄)₂SO₄, 67 mM Tris-HCl (pH = 8.8), 0.01% Tween-20, 2.5 mM MgCl₂], 0.1 mM each dNTP, 5 pM of each primer and 0.6 U Taq polymerase (Pharmacia) in a final volume of 15 μl. All microsatellite forward-primers were 5'-labelled with FAM fluorochrome (Perkin-Elmer). Cycling conditions included an initial denaturation at 94°C for 5 min and pairing at 65°C for 5 min, followed by 30 cycles at 94°C for 45 sec; 60°C with primer pairs DXS1068.PCR1 and AFM150xf10 (or 65°C with primer pairs AFM276xf5 and AFM199wc7) for 45 sec; 72°C for 45 sec, and a final extension period of 20 min at 72°C.

Amplified products were run in a MEGABACE 1000 automatic DNA sequencer with 10× LPA buffer and MegaBACE™ Long Read Matrix with the GT Dye set 2 ET-ROX, FAM (Applied Biosystems) and analyzed with Genetic Profiler^® v.2.0 (Amersham, 2002).

X-inactivation patterns were analyzed in the proband, her mother and in 35 cell hybrid lines previously shown to contain two or more X-chromosome haplotypes. The methylation status of the X chromosome was assayed in a region of the human androgen receptor (AR) gene located in Xq11-q12. This AR region corresponded to nucleotides 451-661 of the HUMANDREC sequence 49 (GenBank M20132), containing restriction sites for the methylation-sensitive endonucleases HhaI and HpaII and a highly polymorphic VNTR. Polymorphisms were initially detected in undigested genomic DNA samples with a 5' FAM-labelled, forward AR-C primer, under specific PCR conditions 50 . X chromosome methylation was assayed by separate digestions of genomic DNA with excess of HhaI and HpaII in final volumes of 22.5 μl at 37°C for 18 hours. Digestion products were subsequently used in PCR reactions, under identical conditions with those used for detecting VNTR polymorphisms. All PCR products were analyzed with Genetic Profiler^®, version 2.0.

The X pentasomy was also demonstrated by FISH with an X whole chromosome probe. The number of X chromosomes present in cell hybrid lines 1PXKA1, 1PXKA3, 1PXKB2, 1PXKF4, 1PXKH2, 1PXKJ6, 1PXKM1, 1PXKQ3 and 1PXKS3 was estimated in 100 cells per cell line. In all cell hybrid lines (except for 1PXKJ6), more than one X haplotype was identified, which were present in different combinations (M1-M2, M1-P, M2-P, MR-P and M1-M2-P).

We used a human specific, X chromosome probe (WCPX) provided by Roscoe Stanyon (Frederick Cancer Research Facility, NCI, NIH), which was amplified and labelled by random priming with the 6MW primer 5'-CCG ACT CGA GNN NNN NAT GTG G-3' (N = any base). Reactions were carried out with 150 ng of probe DNA, 20 pmol of primer, 5 μl of 10× reaction buffer (245 mM TAPs, 500 mM KCl, 20 mM MgCl₂, 2 mM dithiothreitol; pH = 9.3); 0.5 mM dCTP, dATP, dGTP, 0.25 mM dTTP; 5 μl of W-1 detergent (stock solution = 100 μl of W-1 in 10 ml of distilled water), 2 nM FITC-12-dUTP (Roche), and 3 U of Taq Platinum ^® (Invitrogen), in final volumes of 50 μL. Cycling conditions consisted of an initial denaturation at 94°C for 3 min followed by 35 cycles at 94°C for 1 min, a 62°C for 1 min, 72°C for 1 min, and a final extension period at 72°C for 9 min.

Slides were denatured in 70% formamide/2× SSC (pH = 7.0) at 73°C for 5 min and dehydrated in 70%, 85% and 100% ethanol and allowed to dry at room temperature. Probe preparation, incubation with slides and further processing were carried out according to Vysis^® standard protocols for hybridization with whole chromosome probes. Preparations were analyzed with epifluorescence microscopes (Olympus DX-60 and DX-50). Images were captured with QUIPS - PathVysion (Vysis^®) and Applied Imaging.

Analysis of a 380 pb minimal region of the XIST promoter was carried for characterising position - 43 with respect to the possibility of finding C(- 43)A or C(- 43)G in the proband, her mother and in ten cell cell hybrid lines derived from the proband (1PXKA1, 1PXKB1, 1PXKB2, 1PXKB5, 1PXKC1, 1PXKE3, 1PXKG4, 1PXKJ6, 1PXKP3, 1PXKQ2). DNA was amplified with a forward primer 5'-TGAGAACTGGAAAACCCATTG-3' and a reverse primer 5'-ATACGCCATAAAGGGTGTTGG-3'. Reactions were carried out with 200 ng of DNA, 25 pmol of each primer, 0.2 mM of each dNTP and 0.2 U of Taq Platinum^® (Invitrogen), with 1× enzyme buffer and 1.5 mM MgCl₂, in a final volumes of 50 μL. Cycling conditions included an initial denaturation step at 94°C for 5 min, followed by 35 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 45 sec, with a final extension period at 72°C for 15 min in a GeneAmp PCR System 2400 (Applied Biosystems). Amplified products were purified with GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) and eluted in 50 μL of water. Approximately 100 ng of the purified fraction was labelled with 5 pmol of either primer according to the conditions outlined by the DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBace DNA Analysis Systems (GE Healthcare). Sequencing was carried out in a MEGABACE 1000 automatic DNA sequencer. Sequences were aligned manually with Chromas version 1.45 51 and MEGA 4.0 52 .