X Chromosome–Inactivation Patterns of 1,005 Phenotypically Unaffected Females
2006; Elsevier BV; Volume: 79; Issue: 3 Linguagem: Inglês
10.1086/507565
ISSN1537-6605
AutoresJames Amos‐Landgraf, Amy A. Cottle, Robert M. Plenge, Mike Friez, Charles E. Schwartz, John Longshore, Huntington F. Willard,
Tópico(s)Gender Studies in Language
ResumoX-chromosome inactivation is widely believed to be random in early female development and to result in a mosaic distribution of cells, approximately half with the paternally derived X chromosome inactive and half with the maternally derived X chromosome inactive. Significant departures from such a random pattern are hallmarks of a variety of clinical states, including being carriers for severe X-linked diseases or X-chromosome cytogenetic abnormalities. To evaluate the significance of skewed patterns of X inactivation, we examined patterns of X inactivation in a population of >1,000 phenotypically unaffected females. The data demonstrate that only a very small proportion of unaffected females show significantly skewed inactivation, especially during the neonatal period. By comparison with this data set, the degree of skewed inactivation in a given individual can now be quantified and evaluated for its potential clinical significance. X-chromosome inactivation is widely believed to be random in early female development and to result in a mosaic distribution of cells, approximately half with the paternally derived X chromosome inactive and half with the maternally derived X chromosome inactive. Significant departures from such a random pattern are hallmarks of a variety of clinical states, including being carriers for severe X-linked diseases or X-chromosome cytogenetic abnormalities. To evaluate the significance of skewed patterns of X inactivation, we examined patterns of X inactivation in a population of >1,000 phenotypically unaffected females. The data demonstrate that only a very small proportion of unaffected females show significantly skewed inactivation, especially during the neonatal period. By comparison with this data set, the degree of skewed inactivation in a given individual can now be quantified and evaluated for its potential clinical significance. In female mammals, most genes on all X chromosomes in excess of one per autosomal complement are silenced epigenetically early in development, thereby making female X-chromosome gene dosage largely equivalent to that of males.1Lyon MF Gene action in the X-chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2457) Google Scholar, 2Carrel L Willard HF X inactivation profile reveals extensive variability in X-linked gene expression.Nature. 2005; 434: 400-404Crossref PubMed Scopus (1351) Google Scholar The choice of which X chromosome remains active in each cell is thought to be random, so that females have varying X-inactivation ratios, defined as the proportions of cells expressing alleles from one or the other X chromosome. The X-inactivation ratios of females can range from a highly skewed ratio of 0:100, where the same X chromosome is active for all cells, to a 50:50 ratio, where each X chromosome is active in an equal number of cells.3Nance WE Genetic tests with a sex-linked marker: glucose-6-phosphate dehydrogenase.Cold Spring Harbor Symp Quant Biol. 1964; 29: 415-425Crossref PubMed Scopus (50) Google Scholar In unaffected females, the X-inactivation ratio may be of no clinical importance, but a highly skewed X-inactivation ratio may be indicative of carrier status for many X-chromosome disorders, including both cytogenetic abnormalities and certain X-linked Mendelian conditions.4Brown CJ Robinson WP The causes and consequences of random and non-random X chromosome inactivation in humans.Clin Genet. 2000; 58: 353-363Crossref PubMed Scopus (100) Google Scholar, 5Willard HF The sex chromosomes and X chromosome inactivation.in: Scriver CR Beaudet AL Sly WS Valle D Childs B Vogelstein B The metabolic and molecular bases of inherited disease. 8th ed. McGraw-Hill, New York2000: 1191-1221Google Scholar, 6Van den Veyver IB Skewed X inactivation in X-linked disorders.Semin Reprod Med. 2001; 19: 183-191Crossref PubMed Scopus (86) Google Scholar Skewed X-inactivation patterns may occur either as a result of secondary cell selection during development or by primary stochastic or genetic processes.4Brown CJ Robinson WP The causes and consequences of random and non-random X chromosome inactivation in humans.Clin Genet. 2000; 58: 353-363Crossref PubMed Scopus (100) Google Scholar, 7Belmont JW Genetic control of X inactivation and processes leading to X-inactivation skewing.Am J Hum Genet. 1996; 58: 1101-1108PubMed Google Scholar, 8Puck JM Willard HF X inactivation in females with X-linked disease.N Engl J Med. 1998; 338: 325-328Crossref PubMed Scopus (153) Google Scholar The most common reason for highly skewed X inactivation is postinactivation cell selection due to an X-chromosome abnormality that affects cell proliferation either in all cells in the embryo or in a tissue-specific manner. For example, females who carry balanced X;autosome translocations typically exhibit skewed X inactivation because of selection in favor of cells in which the normal X is inactivated.9Mattei MG Mattei JF Vidal I Giraud F Structural anomalies of the X chromosome and inactivation center.Hum Genet. 1981; 56: 401-408Crossref PubMed Scopus (63) Google Scholar, 10Schmidt M Du Sart D Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced X-autosome translocations: a review of 122 cases.Am J Med Genet. 1992; 42: 161-169Crossref PubMed Scopus (129) Google Scholar X-linked disorders are generally rare in females and are usually attributable to advantageous silencing of the X chromosome that carries the mutant allele.5Willard HF The sex chromosomes and X chromosome inactivation.in: Scriver CR Beaudet AL Sly WS Valle D Childs B Vogelstein B The metabolic and molecular bases of inherited disease. 8th ed. McGraw-Hill, New York2000: 1191-1221Google Scholar The presumption is that the initial X-inactivation choice is random but that, during proliferation, cells that have chosen the mutant X chromosome to be active have a significant or total growth disadvantage and are thus underrepresented in the adult carrier. This postinactivation cell selection has been documented in carriers for a number of X-linked diseases.11Nyhan WL Bakay B Connor JD Marks JF Keele DK Hemizygous expression of glucose-6-phosphate dehydrogenase in erythrocytes of heterozygotes for the Lesch-Nyhan syndrome.Proc Natl Acad Sci USA. 1970; 65: 214-218Crossref PubMed Scopus (119) Google Scholar, 12Conley ME Brown P Pickard AR Buckley RH Miller DS Raskind WH Singer JW Fialkow JJ Expression of the gene defect in X-Linked agammaglobulinemia.N Engl J Med. 1986; 315: 564-567Crossref PubMed Scopus (140) Google Scholar, 13Goodship J Carter J Espanol T Boyd Y Malcolm S Levinsky R Carrier detection in Wiskott-Aldrich syndrome: combined use of M27 β for X inactivation studies and as a linked probe.Blood. 1991; 77: 2677-2681PubMed Google Scholar, 14Plenge RM Tranebjaerg L Jensen PKA Schwartz CE Willard HF Evidence that mutations in the X-linked DDP gene cause incompletely penetrant and variable skewed X inactivation.Am J Hum Genet. 1999; 64: 759-767Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 15Plenge RM Stevenson RA Lubs HA Schwartz CE Willard HF Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders.Am J Hum Genet. 2002; 71: 168-173Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar In contrast to negative cell selection, the positive growth advantage of cells in which the mutant X is primarily active has been reported in a few conditions.16Migeon BR Moser H Moser A Axelman J Sillence D Norum R Adrenoleukodystrophy: evidence for X linkage, inactivation, and selection favoring the mutant allele in heterozygous cells.Proc Natl Acad Sci USA. 1981; 78: 5066-5070Crossref PubMed Scopus (225) Google Scholar, 17Josten KM Tooze JA Borthwick-Clarke C Gordon-Smith EC Rutherford TR Acquired aplastic anemia and paroxysmal nocturnal hemoglobinuria: studies on clonality.Blood. 1991; 78: 3162-3167PubMed Google Scholar It has long been recognized from a theoretical perspective that an additional explanation for nonrandom X-inactivation ratios might include mutations in the X-inactivation process itself, which causes one chromosome to be chosen over another at the time of X inactivation in the early embryo.8Puck JM Willard HF X inactivation in females with X-linked disease.N Engl J Med. 1998; 338: 325-328Crossref PubMed Scopus (153) Google Scholar, 18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar Mutations in the X-inactivation pathway are thought to be rare, but studies of individuals with such mutations may provide much information regarding the regulation of the X-inactivation pathway. Studies of large families19Naumova AK Olien L Bird LM Smith M Verner AE Leppert M Morgan K Sapienza C Genetic mapping of X-linked loci involved in skewing of X chromosome inactivation in the human.Eur J Hum Genet. 1998; 6: 552-562Crossref PubMed Scopus (61) Google Scholar, 20Naumova AK Plenge RM Bird LM Leppert M Morgan K Willard HF Sapienza C Heritability of X chromosome-inactivation phenotype in a large family.Am J Hum Genet. 1996; 58: 1111-1119PubMed Google Scholar and females who express X-linked disorders18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar have revealed that a locus or loci on the X chromosome can affect the X-inactivation ratio. Studies of females with Duchenne muscular dystrophy (MIM 300377) also suggest that other modifying loci exist that affect the X-inactivation ratio.21Azofeifa J Voit T Hubner C Cremer M X-chromosome methylation in manifesting and healthy carriers of dystrophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as a cause of the affected phenotypes.Hum Genet. 1995; 96: 167-176Crossref PubMed Scopus (62) Google Scholar, 22Pegoraro E Whitaker J Mowery-Rushton P Surti U Lanasa M Hoffman EP Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.Am J Hum Genet. 1997; 61: 160-170Abstract Full Text PDF PubMed Scopus (106) Google Scholar These limited studies of human families are intriguing, since the X-inactivation ratio is known to be under primary genetic control in the mouse.23Cattanach BM Isaacson JH Controlling elements in the mouse X chromsome.Genetics. 1967; 57: 331-346PubMed Google Scholar, 24Percec I Plenge RM Nadeau JH Bartolomei MS Willard HF Autosomal dominant mutations affecting X inactivation choice in the mouse.Science. 2002; 296: 1136-1139Crossref PubMed Scopus (50) Google Scholar, 25Chadwick LH Willard HF Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous mice.Mammal Genome. 2005; 16: 691-699Crossref PubMed Scopus (24) Google Scholar The major locus controlling the preferential choice of which X chromosome is to remain active in the mouse is the X-linked X-controlling element (Xce) locus, although the mechanism responsible for this effect remains elusive. It has been suggested that similar XCE alleles might exist in the human population.19Naumova AK Olien L Bird LM Smith M Verner AE Leppert M Morgan K Sapienza C Genetic mapping of X-linked loci involved in skewing of X chromosome inactivation in the human.Eur J Hum Genet. 1998; 6: 552-562Crossref PubMed Scopus (61) Google Scholar Whereas the probability that highly skewed X-inactivation ratios occur by chance may be low,3Nance WE Genetic tests with a sex-linked marker: glucose-6-phosphate dehydrogenase.Cold Spring Harbor Symp Quant Biol. 1964; 29: 415-425Crossref PubMed Scopus (50) Google Scholar an accurate assessment of the X-inactivation variance is needed to quantify this probability. With a highly accurate measurement of X-inactivation in a large population, the number of cells present in the embryo when the choice of which X remains active can be determined.26Fialkow PJ Primordial cell pool size and lineage relationships of five human cell types.Ann Hum Genet. 1973; 37: 39-48Crossref PubMed Scopus (124) Google Scholar Previous studies to determine this number used distributions of X-inactivation ratios from apparently unaffected adult females.26Fialkow PJ Primordial cell pool size and lineage relationships of five human cell types.Ann Hum Genet. 1973; 37: 39-48Crossref PubMed Scopus (124) Google Scholar, 27Puck JM Stewart CC Nussbaum RL Maximum-likelihood analysis of human T-cell X chromosome inactivation patterns: normal women versus carriers of X-linked severe combined immunodeficiency.Am J Hum Genet. 1992; 50: 742-748PubMed Google Scholar, 28Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar However, since skewing increases with age in unaffected adult and elderly females,29Fey MF Liechti-Gallati S von Rohr A Borisch B Theilkas L Schneider V Oestreicher M Nagel S Ziemiecki A Tobler A Clonality and X-inactivation patterns in hematopoietic cell populations detected by the highly informative M27β DNA probe.Blood. 1994; 83: 931-938PubMed Google Scholar, 30Busque L Mio R Mattioli J Brais E Blais N Lalonde Y Maragh M Gilliland DG Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age.Blood. 1996; 88: 59-65PubMed Google Scholar, 31Gale RE Fielding AK Harrison CN Linch DC Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.Br J Haematol. 1997; 98: 512-519Crossref PubMed Scopus (187) Google Scholar, 32Sandovici I Naumova AK Leppert M Linares Y Sapienza C A longitudinal study of X inactivation ratio in human females.Hum Genet. 2004; 115: 387-392Crossref PubMed Scopus (44) Google Scholar use of these distributions to predict stem-cell number may not be appropriate, since the predicted stem-cell number is inversely proportional to the distribution variance.26Fialkow PJ Primordial cell pool size and lineage relationships of five human cell types.Ann Hum Genet. 1973; 37: 39-48Crossref PubMed Scopus (124) Google Scholar In addition, any errors in determining the X-inactivation ratio, likely dependent on the particular assay being used, would influence and likely increase the apparent variance. X-inactivation ratios have been determined with a variety of assays that rely on the differential expression of polymorphic X-linked genes or, less directly, on the differential methylation of sequences on the active and inactive chromosomes.26Fialkow PJ Primordial cell pool size and lineage relationships of five human cell types.Ann Hum Genet. 1973; 37: 39-48Crossref PubMed Scopus (124) Google Scholar, 28Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar, 29Fey MF Liechti-Gallati S von Rohr A Borisch B Theilkas L Schneider V Oestreicher M Nagel S Ziemiecki A Tobler A Clonality and X-inactivation patterns in hematopoietic cell populations detected by the highly informative M27β DNA probe.Blood. 1994; 83: 931-938PubMed Google Scholar, 33Gandini E Gartler SM Glucose-6-phosphate dehydrogenase mosaicism for studying the development of blood cell precursors.Nature. 1969; 224: 599-600Crossref PubMed Scopus (10) Google Scholar, 34Vogelstein B Fearon ER Hamilton SR Preisinger AC Willard HF Michelson AM Riggs AD Orkin SH Clonal analysis using recombinant DNA probes from the X-chromosome.Cancer Res. 1987; 47: 4806-4813PubMed Google Scholar, 35Rupert JL Brown CJ Willard HF Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.Eur J Hum Genet. 1995; 3: 333-343PubMed Google Scholar Because of its high polymorphism content, the most commonly used assay examines differential methylation of the human androgen receptor (AR) gene.28Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar Although the assay has been used in many studies, a wide range of results has been reported with respect to the distribution of X-inactivation ratios in the population(s) under study,18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar, 20Naumova AK Plenge RM Bird LM Leppert M Morgan K Willard HF Sapienza C Heritability of X chromosome-inactivation phenotype in a large family.Am J Hum Genet. 1996; 58: 1111-1119PubMed Google Scholar, 28Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar, 32Sandovici I Naumova AK Leppert M Linares Y Sapienza C A longitudinal study of X inactivation ratio in human females.Hum Genet. 2004; 115: 387-392Crossref PubMed Scopus (44) Google Scholar,36Gale RE Mein CA Linch DC Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay.Leukemia. 1996; 10: 362-367PubMed Google Scholar, 37Sharp A Robinson D Jacobs P Age- and tissue-specific variation of X chromosome inactivation ratios in normal women.Hum Genet. 2000; 107: 343-349Crossref PubMed Scopus (262) Google Scholar, 38Karasawa M Tsukamoto N Yamane A Okamoto K Maehara T Yokohama A Nojima Y Omine M Analysis of the distribution of CAG repeats and X-chromosome inactivation status of HUMARA gene in healthy female subjects using improved fluorescence-based assay.Int J Hematol. 2001; 74: 281-286Crossref PubMed Scopus (15) Google Scholar, 39Beever CL Stephenson MD Peñaherrera MS Jiang RH Kalousek DK Hayden M Field L Brown CJ Robinson WP Skewed X-chromosome inactivation is associated with trisomy in women ascertained on the basis of recurrent spontaneous abortion or chromosomally abnormal pregnancies.Am J Hum Genet. 2003; 72: 399-407Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar with as many as 20%–30% of the population reported to show highly skewed ratios. Although this presumably reflects, in part, the relatively small control groups reported, it may also reflect assay variation; reproducibility, in particular, is a key parameter for an assay that is highly sensitive to partial restriction-enzyme digestion and to potential PCR quantitation or electrophoresis artifacts. A definitive data set of X-inactivation ratios in a large sample of phenotypically unaffected females from the general population would be useful as a baseline for studies of X inactivation in various patient populations and/or genetically defined cohorts. Toward this goal, we have determined the distribution of X-inactivation patterns in blood samples from >1,000 newborn infants and adult women, using the AR X-inactivation assay. The results provide a comprehensive estimate of the frequency of individuals in the general population with highly skewed X-inactivation ratios and suggest that screening of the newborn female population may be an effective means of ascertaining individuals at increased risk of carrying some deleterious X-linked defects. A total of 1,284 anonymized DNA samples from either adult peripheral or newborn cord blood were obtained from several sources. All samples were obtained from phenotypically unaffected individuals from the general control population or from family studies in which it was determined that the individuals did not carry the specific defect in question. The racial and ethnic composition of the sample population was mixed. The following female samples were tested: 127 from the general adult population of Cleveland (provided by Drs. Stuart Schwartz and Georgia Wiesner, Case Western Reserve University), 77 from Botnia, Finland40Mahtani MM Widen E Lehto M Thomas J McCarthy M Brayer J Bryant B Chan G Daly M Forsblom C Kanninen T Kirby A Kruglyak L Munnelly K Parkkonen M Reeve-Daly MP Weaver A Brettin T Duyk G Lander ES Groop LC Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families.Nat Genet. 1996; 14: 90-94Crossref PubMed Scopus (320) Google Scholar (provided by Drs. Melanie Mahtani and Eric Lander), 106 from a Mennonite group in western Pennsylvania (provided by Dr. Eric Puffenberger), 105 from Ashkenazi Jewish mothers with normal pregnancies (provided by Dr. David Gurwitz, National Laboratory for the Genetics of Israeli Populations, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv). The remaining blood samples were from newborn cord or from mothers of newborns from a six-county area in the upper Savannah region of South Carolina. All samples were obtained with approval from the respective institutions and were fully anonymized before transfer to Case Western Reserve University for X-inactivation analysis. To determine the X-inactivation pattern, we examined the methylation status of the AR locus, as described elsewhere.18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar Briefly, ∼50–150 ng of DNA was digested in duplicate for 16 h either with HpaII plus RsaI or with RsaI alone as a control. In addition, digests with HhaI and RsaI, as well as CfoI and RsaI, were run on a select number of samples to test the accuracy of the assay. After digestion, the enzymes were inactivated at 80°C for 20 min, and one-tenth of the resulting digest was used for PCR amplification as described elsewhere.18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar The resulting products were run on an ABI 373A automated sequencer or an ABI 3100 genetic analyzer and were analyzed by GeneScan software (Applied Biosystems). The raw peak height values of the digested samples were corrected for amplification efficiency by using the average of the two samples digested with RsaI alone (a restriction enzyme with sites outside the PCR product, which is not sensitive to methylation and thus digests active and inactive X alleles equally). The X-inactivation pattern (expressed arbitrarily as a ratio of the smaller:larger allele) is the resulting corrected average of the duplicate samples digested with HhaI and RsaI. Samples were scored only if the two alleles were separated by more than two CAG repeats, to avoid uncertain contributions of one peak to the other. In addition, one control male sample and one sample from a female known from an earlier study18Plenge RM Hendrich BD Schwartz C Arena JF Naumova A Sapienza C Winter RM Willard HF A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.Nat Genet. 1997; 17: 353-356Crossref PubMed Scopus (217) Google Scholar to have a completely nonrandom pattern of X inactivation (>98:2) were included in every batch of samples, to control for complete digestion and amplification efficiency. Transformed lymphoblast cell lines were grown and harvested for DNA and RNA, as described elsewhere.35Rupert JL Brown CJ Willard HF Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.Eur J Hum Genet. 1995; 3: 333-343PubMed Google Scholar RNA was isolated with the Stratagene Strataprep Total RNA Miniprep Kit. cDNA was prepared by using plus and minus reverse-transcriptase reactions, to control for DNA contamination. The resulting cDNA was PCR-amplified with primers designed to detect an HinfI restriction-enzyme polymorphism in exon 6 of the XIST gene, followed by a single extension reaction with a fluorescently labeled primer.35Rupert JL Brown CJ Willard HF Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.Eur J Hum Genet. 1995; 3: 333-343PubMed Google Scholar The resulting product was digested with HinfI for at least 4 h at 37°C, and the products were run on an ABI 373A automated sequencer. Peak height values of the resulting raw data were analyzed with GeneScan software, as described above. Control samples for complete digestion of the amplified products were included for every gel. Because of the variability in the reports of distribution of X-inactivation patterns determined by use of the AR assay, we examined some of the variables that might influence the apparent results. We began by addressing which methylation-sensitive restriction enzyme yielded the most-consistent and reproducible results for us. Triplicate samples were assayed by using the restriction enzyme RsaI in conjunction with a methylation-sensitive restriction enzyme—HpaII, HhaI, or CfoI (an isoschizomer of HhaI), each of which has sites within the AR PCR product—as well as a mock-digested control with use of RsaI alone. HpaII and HhaI results were highly correlated with each other, but CfoI consistently generated more variable results (>10% variance). On further investigation, CfoI was found to be only partially heat-inactivated (Boehringer Mannheim and Gibco BRL) and could be completely inactivated only after phenol/chloroform extraction. Presumably, the resulting digested product continued to be digested during PCR amplification and yielded much more variable results between duplicates. To increase the level of precision and accuracy in this data set, we therefore used only HpaII as the methylation-sensitive restriction enzyme, in conjunction with RsaI for data generation. Reproducibility of the data set was confirmed by assaying all samples in duplicate and by reassaying 120 samples chosen at random with HhaI as the methylation-sensitive enzyme (for paired HpaII- and HhaI-digested samples, P>.1 [Student's t test]; r=0.97 [Pearson correlation coefficient]). To determine, in a quantitative manner, whether the X-inactivation patterns measured indirectly at the AR locus correlate with X-inactivation patterns determined directly by gene expression, we also examined a transcribed polymorphism at the XIST locus. Since XIST is expressed exclusively from the inactive X chromosome in female somatic cells,41Brown CJ Ballabio A Rupert JL Lafreniere RG Grompe M Tonlorenzi R Willard HF A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.Nature. 1991; 349: 38-44Crossref PubMed Scopus (1081) Google Scholar this provides a direct estimate of the proportion of cells carrying a particular XIST allele on the inactive X chromosome.35Rupert JL Brown CJ Willard HF Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.Eur J Hum Genet. 1995; 3: 333-343PubMed Google Scholar We determined the X-inactivation patterns of 18 polyclonal lymphoblast cell lines informative at both the AR and XIST loci, using both assays. Because the phase of the AR and XIST alleles was not known, we used a best-fit model to correlate the data. Whereas such a model is insensitive to allelic phase at X-inactivation patterns near 50:50, it is highly sensitive at more-extreme patterns, as frequently observed in clonal or oligoclonal lymphoblast lines.35Rupert JL Brown CJ Willard HF Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.Eur J Hum Genet. 1995; 3: 333-343PubMed Google Scholar On the basis of this model, X-inactivation patterns determined by the AR methylation assay were highly correlated with those determined directly by XIST expression (R2 = 0.969), which indicates that the AR assay is an accurate measure of X inactivation (data not shown). Notwithstanding the apparent robustness of the assay, we do note what appears to be a modest shift toward the left in the X-inactivation distributions (see, e.g., fig. 1), consistent with a slight bias toward amplification of the smaller AR allele. This may reflect a slight overcorrection when the RsaI digest is used; nonetheless, this shift is not statistically significant and has not been explored further. The principal advantage of the AR assay for measuring X-inactivation patterns is the high heterozygosity of a (CAG)n polymorphism near the 5′ end.28Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar Indeed, only ∼8% of the 1,284 samples were uninformative for this polymorphism and were thus excluded from the study. However, because of the stutter bands frequently observed in minisatellite repeats and to avoid potential artifacts that might interfere with quantitation, we also rejected any heterozygous samples in which the stutter band from the larger a
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