ABSTRACT A 46,X,r(X) karyotype was found in a three and a half year old girl with short stature, facial dysmorphism and developmental delay. The clinical findings were consistent with the

phenotype described in a limited number of patients with small ring X chromosomes lacking the _XIST_ locus, a critical player in the process of X chromosome inactivation. Surprisingly, in

our patient, fluorescent _in situ_ hybridisation demonstrated that the _XIST_ locus was present on the ring X. However, expression studies showed that there was no _XIST_ transcript in

peripheral blood cells, suggesting that the ring X had not been inactivated. This was confirmed by the demonstration that both of the patient's alleles for the androgen receptor gene

between _DXS8037_ and _XIST_ and one in the short arm in Xp11.2 between _DXS1126_ and _DXS991_. To attempt to determine why the _XIST_ gene failed to be expressed, the promoter region was

sequenced and found to have a base change at the same location as a variant previously associated with nonrandom X chromosome inactivation. This mutation was not seen in over one hundred

normal X chromosomes examined; however, it was observed in the paternal grandmother who did not show substantial skewing of X chromosome inactivation. SIMILAR CONTENT BEING VIEWED BY OTHERS

A PATERNALLY INHERITED 1.4 KB DELETION OF THE 11P15.5 IMPRINTING CENTER 2 IS ASSOCIATED WITH A MILD FAMILIAL SILVER–RUSSELL SYNDROME PHENOTYPE Article 11 November 2020 DISTAL 2Q DUPLICATION

IN A PATIENT WITH INTELLECTUAL DISABILITY Article Open access 10 November 2022 CYTOGENOMIC CHARACTERIZATION OF MOSAIC X-RING CHROMOSOMES IN SEVENTEEN PATIENTS WITH TURNER SYNDROME (TS)-42 

YEARS OF EXPERIENCE AT A SINGLE-SITE INSTITUTION Article Open access 14 April 2025 INTRODUCTION Ring X chromosomes have been recognised in girls with Turner syndrome, often with mosaicism

for a 45,X cell line. In some instances, they are associated with mental retardation and a distinct phenotype of short stature, facial dysmorphism characterised by long palpebral fissures, a

relatively broad nasal root and tip, anteverted nares, a wide mouth with a thin upper lip, soft tissue syndactyly, and mental handicap. Careful cytogenetic characterisation of the ring X

chromosomes has suggested that the smaller the size of the ring X, the more likely are the findings of mental handicap and dysmorphic features in the patient. This has been attributed to

failure of dosage compensation, by X chromosome inactivation, for the genes on the ring chromosome.1,2,3 A region of the proximal long arm of the X chromosome is required in _cis_ for

inactivation of the chromosome,4,5 and contains the _XIST_ gene, which is expressed exclusively from the inactive X chromosome6 and is necessary for X inactivation in mice (reviewed in Avner

and Heard7). Smaller ring chromosomes may lack the _XIST_ locus, rendering them functionally disomic for the genes present on the ring.8,9 The phenotype of individuals with small ring X

chromosomes presumably results from the continued expression of genes in the pericentromeric region of the X chromosome due to failure of inactivation.10 In females, the presence of

mosaicism for a ring X lacking _XIST_ has been correlated with mental retardation, facial dysmorphism often described as similar to the facies of Nikkawa syndrome, limb anomalies and

abnormal pigmentation.8,11,12,13,14,15,16,17 Even more severe phenotypes have been reported, including prune-belly syndrome in a stillborn fetus with a ring X lacking _XIST_18 and

anencephaly and diaphragmatic hernia in a female fetus with a ring X that was not characterised for _XIST_ expression.19 In addition, there have been reports of males with a supernumary ring

X chromosome, also lacking _XIST_, in some cases with a somewhat similar phenotype, including learning disabilities or mental handicap of varying severity, facial dysmorphism and digital

anomalies.8,20,21,22 There are rare cases where a small ring containing the _XIST_ gene has been observed associated with the ring X phenotype. Many of these patients lack expression of the

inactive X-specific transcript, which presumably precludes inactivation of the chromosome.9 We now report the characterisation of an additional case of a small ring X chromosome which

includes the _XIST_ locus but fails to express the gene. A mutation at a site previously associated with nonrandom X-chromosome inactivation was identified in the girl; however, the same

change was found in her paternal grandmother who did not show substantial skewing of X-chromosome inactivation. PATIENT AND METHODS PATIENT This girl presented with dysmorphic features,

short stature and developmental delay. She was the first child of healthy nonconsanguineous, young, white parents. She was born at 37 weeks of gestation following an uncomplicated pregnancy.

Birth weight was 2895 g (>25th percentile). Her neonatal course was complicated by hypoglycemia with no recognised specific cause. This resolved spontaneously. Milestones were somewhat

delayed. She walked at 16 months, had a pincer grasp at 2 years, and used two to three word phrases by 3 years. When assessed using the Bailey Scales of Infant Development at 3 years, 7

months, level of function approximated that of a child of 2 years, 3 months. At 5.5 years, language skills were significantly delayed. She did not consistently recognise colors or letters of

the alphabet. Social skills were immature. She was not able to pedal a tricycle. When examined at 3.5 years, height of 89 cm and weight of 12.8 kg were just above the 3rd percentile. OFC of

50.5 cm was between the 50th and 75th percentiles. Inner canthal distance of 3 cm was proportionate to the OFC. Total hand and middle finger lengths were below the 3rd percentile,

consistent with the clinical impression of brachydactyly. Facial appearance was distinctly dysmorphic (Figure 1). It was slightly asymmetric with left eye position lower than the right, and

there was mild malar flattening. Ears were normal. Alveolar ridges were moderately thickened, although teeth were normal. Philtrum was poorly defined. Lower jaw was normal, although in

infancy it had been described as being small. Hair pattern and neck shape were normal, as was chest shape. There was a clinical impression of short limbs but she did not cooperate with

measuring this. Mild soft tissue syndactyly of right middle and index finger was seen, and there was significant soft cutaneous syndactyly of left 4th and 5th toes. One large diffuse café au

lait spot was seen on the right buttock, and a small one was present on the right knee. Otherwise, the examination was unremarkable. Investigations showed a normal brain CT scan.

Brachydactyly was seen on a radiograph of the upper limb at 9 months of age, but no other bony anomalies were noted. Renal ultrasound was normal at age 4 years of age, but a hypoplastic

uterus was described. Echocardiogram was normal. TSH was normal. Cytogenetic testing was done at 3.5 and 4.5 years of age. CYTOGENETIC METHODS Peripheral blood specimens were set up for

chromosomal analysis using routine methods, and metaphases were examined with GTG-banding.23 Fluorescent _in situ_ hybridisation (FISH) was carried out according to supplier's protocol

(Oncor, Gaithersburg, MD, USA), using probes for centromeric alphoid DNA sequences from X (_DXZ1_) or Y (_DYZ3_) chromosomes, and for the _XIST_ locus. Fixed cells were saved for preparation

of RNA to evaluate expression of the _XIST_ gene. MOLECULAR METHODS DNA was prepared by salt/SDS precipitation,24 while RNA was prepared by acid-guanidinium extraction.25 DNA and RNA was

isolated from cells stored in fixative after cytogenetic analysis, blood, or cultured control cell lines, GM07348 (female), GM07033 (male), GM06563 (female with large ring X) and GM07059

(female with non-random X inactivation), obtained from the Coriell Cell Repository. cDNA was synthesised as previously described.6 PCR amplification of DNA and cDNA used primers and

conditions listed in Table 1 with 1 μM primer, 20 μM dNTPs and 10 U Taq polymerase (Gibco-BRL) unless otherwise noted. Methylation of the androgen receptor (_AR_) locus was analysed as has

been previously described.26 For microsatellite analysis, PCR products were resolved on polyacrylamide gels and observed after staining with silver, while other PCR products were resolved on

2% agarose gels stained with ethidium bromide. The promoter region of _XIST_ was amplified from the patient using XIST primers, U2 and H3, and purified product was sequenced using U2 and PM

primers by the NAPS facility, UBC. A primer was designed with a mismatch (PM2, Table 1) to generate a _Ban_I restriction enzyme site in the presence of the −43A variant base. After

amplification with primers PM and PM2, PCR products were digested with 15 U of _Ban_I, and resolved on polyacrylamide gels. In the presence of the −43A, 16 bp were removed from the end of

the 145 bp fragment. To examine methylation in this region, 1 μg of DNA was digested with 15 U of _Hha_I in a 20 μl reaction. The reaction was then heated to 95°C for 5 min and then 1 μl was

amplified with primers PM and PM2. The PCR product was digested with _Ban_I to distinguish between the two alleles. RESULTS CYTOGENETIC ANALYSIS GTG-banding analysis of the initial

peripheral blood specimen showed mosaicism for two cell lines: 28% of the cells had 45,X; 72% had 46 chromosomes including one normal X chromosome and a small ring with bands consistent with

an X chromosome origin (Figure 2A). A second blood sample 13 months later had the ring X chromosome in all metaphases examined. FISH with _DXZ1_ and _DYZ3_ showed that the marker was of X

chromosome origin (not shown). FISH with a DNA probe for the _XIST_ locus demonstrated fluorescent signals on both the normal X and the ring X chromosomes (Figure 2B). The karyotype was

designated 46,X,r(X)(p11.3q13).ish r(X)(XIST+). ACTIVITY OF THE X CHROMOSOMES As shown in Figure 3A, cDNA from the patient was amplified with primers for _PGK1_, but no product was detected

with primers for _XIST_. This result was similar to the amplification observed in males who do not have an inactive X chromosome, while cDNA from female cells with a normal karyotype or

containing a large ring X chromosome (GM06563) showed amplification for _XIST_ as well as the _PGK1_ control gene. The patient was heterozygous for the polymorphic trinucleotide repeat in

the androgen receptor (AR) gene,26 as shown by the presence of two bands in the uncut DNA in Figure 3B. After digestion with the methylation-sensitive restriction enzyme, _Hha_I, DNA from

active (unmethylated) X chromosomes should be digested, eliminating the template for amplification. Amplification of both alleles in the patient was eliminated, suggesting that both alleles

were unmethylated and active. In a control female cell line (GM07059) with nonrandom X-chromosome inactivation, digestion with _Hha_I resulted in loss of only one allele, that from the

active X chromosome (Figure 3B). The _ZXDA_ gene in Xp11 contains a polymorphic CA repeat in the transcribed but untranslated region of the gene. This gene is subject to X-chromosome

inactivation, so expression of only the allele on the active X chromosome should be detected.27 In the patient, expression of both alleles was observed (Figure 3C), although one allele was

fainter in both DNA and cDNA, presumably due to mosaicism for the ring chromosome. MOLECULAR CHARACTERISATION OF THE RING X CHROMOSOME DNA isolated from the mother, the father and the

patient was amplified with primers for polymorphic X-linked markers. Informative markers are shown in Figure 4A. For all loci, the patient retained a maternal allele, whereas paternal

alleles were retained only in the pericentromeric region, demonstrating that the ring X is paternal in origin and spans from a breakpoint between _DXS1126_ and _DXS991_ in Xp11 to a

breakpoint between _DXS8037_ and _XIST_ in Xq13. To examine possible causes for the failure of expression of the _XIST_ locus on the ring X chromosome, the promoter region of _XIST_ in the

patient was sequenced. Only one basepair difference was observed in comparison to previously described _XIST_ minimal promoter sequence.28 As shown in Figure 4B, there was a small ‘A’ peak

beneath the ‘C’ peak at basepair-43 of _XIST_. This change was confirmed by creating a primer with a mismatch (PM2, as shown in Figure 4B) that would generate a _Ban_I restriction site in

the presence of the base change. Amplified product from the patient's DNA and the patient's father's DNA was digested with _Ban_I, suggesting that the variant _XIST_ sequence

was paternally inherited. The band corresponding to the variant sequence (digested product, Figure 4C) was fainter in the patient, consistent with the variant sequence being on the

paternally inherited ring chromosome that is not present in all of her cells. The patient's paternal grandmother was heterozygous for the variant (Figure 4C). Over 100 X chromosomes

from male and female controls were examined for this variant using the _Ban_I assay, and no other individuals were identified with the same change. To determine the effect of this sequence

alteration we examined the X inactivation status of the chromosome carrying the variant. The primers that recognise the variant basepair flank two methylation-sensitive enzyme sites, which

are known to be differentially methylated on the active and inactive X chromosome29 (Figure 5A), making it possible to analyse methylation of the two _XIST_ alleles individually. As shown in

Figure 5B, predigestion with _Hha_I did not reduce the intensity of either the _Ban_I cut (lower=variant) or uncut (upper=normal) alleles, suggesting that both _XIST_ promoter region

alleles are methylated. DNA from the patient's paternal grandmother (carrier, Figure 5B) amplified both alleles after predigestion, demonstrating that both the variant and normal

alleles are unmethylated in a proportion of the grandmother's cells. To confirm the inactivation of each X chromosome in a subset of the grandmother's cells, we examined

methylation at the _AR_ locus. Both alleles were amplified after predigestion, with a slight alteration in intensity from the uncut sample (Figure 5C). Quantitation performed using the NIH

IMAGE program with scanned gels for both _AR_ and _XIST_, suggest that the grandmother has 60% of cells with the variant on the active X chromosome. DISCUSSION Chromosomal analysis of the

patient was initiated because of short stature and mental handicap. The initial chromosome result of 45,X/46,X,r(X)(p11.3q13) suggested the girl's phenotype might be explained by

functional disomy of genes on the small ring X chromosome; however, FISH revealed that the _XIST_ locus was present on the ring. Investigation of the expression of the _XIST_ gene

demonstrated no expression. The activity of the ring X chromosome was confirmed by the lack of methylation at the _AR_ locus and by the expression of both _ZXDA_ alleles. The direct

examination of gene activity on the ring chromosome using the expressed polymorphism in _ZXDA_ should be valuable to resolve conflicts that have occasionally arisen in determining ring X

activity using AR methylation or replication timing.30,31,32 There appears to be a general phenotype associated with active small ring X chromosomes that includes short stature and mental

handicap. The short stature, which is also found in Turner syndrome, may be due to the absence of genes regulating stature such as SHOX (short stature homeobox-containing gene) in the

pseudoautosomal region of the short arm of the X chromosome.33 The observed mental handicap specifically associated with the small active ring X chromosome is likely due to functional disomy

for genes that are normally inactivated. Some of the considerable variation observed between individuals with ring X chromosomes might be related to the parental origin of the ring X,

mosaicism or the size of the ring X chromosome. Parental origin does not seem to correlate with outcome,34 although uniparental disomy (UPD) has been associated with a more severe outcome.30

This could reflect the duplication of an X chromosome after the time at which marking of an X chromosome to remain active occurs, thereby resulting in derivative chromosomes which are

unable to inactivate.35 In the case reported here, the normal X chromosome was from the mother and the ring chromosome was derived from the father, which is not uncommon for rearranged X

chromosomes (rings or isochromosomes).36 Thus, both chromosomes were present throughout early development and would be present at the time of inactivation. Mosaicism is very large

confounding factor in attempting phenotype-genotype correlations to define critical regions for different aspects of the ‘syndrome’. Cytogenetic testing in our patient at different times

showed different percentages of ring chromosome, demonstrating the difficulty in predicting levels of mosaicism from single samples of a single tissue. Rare cases where _XIST_ expression has

been detected with the more severe phenotype generally associated with an active ring chromosome12,30,37 may reflect the presence of active ring (X) chromosome at a critical stage in

development. The extent of DNA missing from the patient's ring chromosome was determined by allelotyping DNA from the patient and her parents. The breakpoint in Xp11.2 was between the

_DXS1126_ and _DXS991_. Four of the five ring X chromosomes reported by Wolff _et al_8 had breakpoints between _UBE1_ and _DXS423E_, an approximately 10 Mb region which would overlap the

region defined for our proband. Other rings retaining the _XIST_ gene, but not expressing it, showed variable Xp breaks9 so the location of the short arm breakpoint does not seem to be

related to inability to express _XIST_. The breakpoint in the long arm in our case occurred between _XIST_ and the nearby _DXS8037_ locus. Both loci are found within a single YAC, suggesting

that they might be less than 1 Mb apart.38 Two of the previous _XIST_-containing ring X chromosomes failing to express _XIST_ also had breakpoints within 1 Mb of _XIST_. The _XIST_ gene is

apparently intact in our patient, as a polymorphism was identified in the promoter that is telomeric to the 3′ end of the gene.39 While it is possible that more distal elements are required

for proper _XIST_ expression, two other _XIST_-positive non-expressing chromosomes contained substantially more distal DNA, even more than other rings expressing _XIST_.9 A sequence change

was detected in the promoter of the XIST gene in the patient at position −43 bp. Intriguingly, this is the same site associated with nonrandom X-chromosome inactivation.40 The change in our

patient was from C to A (C-43A) while the previously reported change was to a G (C-43G). This base is not evolutionarily conserved, as a G is present in the mouse sequence and an A in

rabbits.28 The C-43G variant was detected in two families, with skewing of inactivation ranging from more than 95% inactivation of the chromosome with the variant in several individuals to

only 55% skewing in others. The C-43A variant failed to undergo inactivation in the patient reported here, and was also associated with the active X chromosome in the grandmother, although

she showed only 60 : 40 skewing of inactivation. Furthermore, it has been reported that skewing increases with age.41 Thus, the extent of skewing in this individual is not different from

that seen in the general population. It is, however, clear that these variant sequences are not common in the population. We have examined over 100 X chromosomes using the _Ban_I assay

described, which will detect both the C-43A and C-43G variants, and have not detected any additional cases. The two families were the only instances of the C-43G change in 1 166 independent

chromosomes.40 A recent analysis of 32 females with skewed inactivation also did not detect additional cases.42 Thus, it is surprising that these variants were detected in individuals with

peculiarities in their X inactivation. Variability in the extent of inactivation might reflect interaction with other variable components of the inactivation pathway. It is also possible

that the variability seen for the inactivation of the X chromosome carrying the C-43A variant was due to the chromosome being unable to lose the mark to remain active, in a manner analogous

to imprinting center mutations.43 The _Hha_I assay used to search for the C-43G variant would not detect the C-43A variant, so it will be interesting to examine other females with nonrandom

patterns of X inactivation for this variant. It will also be interesting to examine the promoter sequence of other ring X chromosomes failing to express _XIST_ for this, or other, variants.

In conclusion, we report a new case of a ring X chromosome containing _XIST_, but failing to express it, resulting in the derivative chromosome remaining active. Functional disomy is

presumed to account for the characteristic phenotype of dysmorphic features, brachydactyly and developmental delay. The presence of _XIST_ on the small, active ring X chromosome underscores

the need to combine molecular analyses along with FISH for _XIST_ in the analysis of such chromosomes. Detection of a variant sequence in promoter of _XIST_ at the same position as another

variant associated with nonrandom X chromosome inactivation is intriguing. However, the essentially random inactivation in grandmother with the same variant suggests that, if this change

leads to failure to express _XIST_, the mechanism is complex. ELECTRONIC DATABASE INFORMATION URLs for data in this article are as follows: http://locus.umdnj.edu/higms/ for the Coriell Cell

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to the centre _Nat Genet_ 1996 14: 119–121 Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The family of our patient is thanked for their interest and cooperation. The

assistance of Viola Freemen and Leslie Malloy, Regional Cytogenetics Laboratory, Hamilton Health Sciences Corporation, in preparation of cytogenetic figures is acknowledged. This research

was supported by a research grant from the Medical Research Council of Canada (MOP13690) to CJ Brown. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Medical Genetics, University

of Alberta and Cytogenetics Laboratory, Stollery Children's Hospital, Edmonton, AB, Canada Darrell J Tomkins * Department of Medical Genetics, University of British Columbia,

Vancouver, BC, Canada Helen L McDonald & Carolyn J Brown * Department of Laboratory Medicine, The Credit Valley Hospital, Mississauga, ON, Canada Sandra A Farrell Authors * Darrell J

Tomkins View author publications You can also search for this author inPubMed Google Scholar * Helen L McDonald View author publications You can also search for this author inPubMed Google

Scholar * Sandra A Farrell View author publications You can also search for this author inPubMed Google Scholar * Carolyn J Brown View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Darrell J Tomkins. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Tomkins, D., McDonald,

H., Farrell, S. _et al._ Lack of expression of _XIST_ from a small ring X chromosome containing the _XIST_ locus in a girl with short stature, facial dysmorphism and developmental delay.

_Eur J Hum Genet_ 10, 44–51 (2002). https://doi.org/10.1038/sj.ejhg.5200757 Download citation * Received: 14 August 2001 * Revised: 21 November 2001 * Accepted: 22 November 2001 * Published:

28 February 2002 * Issue Date: 01 January 2002 * DOI: https://doi.org/10.1038/sj.ejhg.5200757 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content:

Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * ring

X * XIST * X chromosome inactivation * dosage compensation * Turner syndrome