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A non-mosaic ring chromosome 9 in a newborn baby referred for ambiguous genitalia: a case study.


The incidence of chromosome ring formation in humans is quite rare, with an estimated frequency of 1 in 50,000 human fetuses (1). The mechanism for this chromosomal abnormality remains unknown at present but it is strongly associated with the varying loss of the telomere ends on each arm of the chromosome. Critically, telomeres confer stability to chromosomes by preventing DNA degradation and end-to-end fusion during replication. Loss of this function results in unstable chromosomes which, in ring chromosome formation, result in annealing of the "sticky ends" thereby preventing further loss of DNA, but resulting in an unbalanced genome.

Normal human chromosomes are linear in structure consisting of two "arms" (designated "p" and "q") that flank a single active centromere. In addition to this, the ends of the chromosomes are "capped" with structures called telomeres, which give the chromosomes stability by preventing degradation of the ends during DNA replication. Rarely, ring chromosomes are formed, which occur at an estimated incidence of 1/50,000 in human fetuses (1). The mechanism of formation is unclear, but involves the loss of varying extents of the telomeric ends of the p and q arms, followed by annealing of the "sticky" ends in order to prevent further loss of genetic material. Although a ring chromosome is considered to have effectively regained its stability, the genome is unbalanced due to the loss of DNA. If this loss encompasses actively transcribed genes, then the resulting monosomy may lead to a phenotypic abnormality.

Ring chromosomes are divided into two groups: those in which one normal chromosomal homologue is replaced by a ring equivalent, and those in which the ring chromosome is additional to the two normal copies of that chromosome; such a ring is termed a supernumary. Furthermore, many reported ring chromosome cases are mosaic in which the individual possesses at least two cell lines of varying chromosomal composition: typically a normal cell line without the ring chromosome, and another with a ring chromosome. In addition, the proportion of these cell lines may vary between tissues, which further complicate the molecular confirmation of a clinical diagnosis and the prognosis.

Only 20 cases of ring chromosome 9 have been reported in the literature (2). Most of the breakpoints in each arm are not highly resolved, so genotype:phenotype classifications are difficult to make. However, some clinical features are common, including short stature, microcephaly, mental retardation and seizures. Less common features include heart defects, ambiguous male genitalia and cleft palate.

The case presented here involved an initial diagnosis of cleft palate, but with ambiguous genitalia; post mortem analysis at 8 weeks of age also identified a heart defect and renal cysts. Conventional and molecular karyotyping led to genetic counseling for the parents and high resolution identification of the extent of the genomic loss on the ring chromosome 9.


Case report

The infant was delivered at 38 weeks gestation after induction of labour because of clinical and sonographic detection of growth deceleration during the last few weeks of pregnancy. At birth, all growth parameters were below the third centile. The baby was admitted to NICU (Neonatal Intensive Care Unit) with hypoglycemia, and feeding difficulties. On examination, he was noted to have a posterior cleft palate, microphallus with hypospadias, with both testes high in the underdeveloped scrotum.

A request for cytogenetic analysis (see below) was based on the clinical presentation of ambiguous genitalia, cleft palate, pre and postnatal growth retardation. Antenatal ultrasound did not detect any major organ anomalies. However, at 8 weeks of age, the infant was admitted to hospital with respiratory distress and later died. The cause of death was cited as heart failure, secondary to patent ductus arteriosus. Subcapsular renal cysts were also noted sonographically at demise.

When the child was one day of age, we received a peripheral blood sample in lithium heparin for routine cytogenetic analysis (conventional karyotyping). Culturing was carried out according to standard protocols. The initial analysis of 30 cells showed a nonmosaic male karyotype with all cells carrying one normal homologue of chromosome 9 and one ring chromosome, 46,XY,r(9)(p24q34). Increasing this analysis to 70 blood lymphocyte metaphases identified a low level of mosaicism with two cells showing a loss of the ring 9 (resulting in monosomy for chromosome 9), one cell with a broken ring 9, and one cell with a double ring resulting in trisomy for chromosome 9 (Figure 2). Subsequent cytogenetic analysis of peripheral blood of the parents showed that they carried two normal copies of chromosome 9; hence the child's ring chromosome was de novo.


Panel A shows the outcomes of sister chromatid exchange (SCE) involving a ring chromosome. An even number of SCEs in the same direction can lead to normal symmetrical segregation of chromatids; an even number of SCEs in different directions can lead to interlocked rings, but only the breakage of the rings will allow them to segregate; an odd number of SCEs can lead to the loss of the ring in one cell, and two parallel chromatids forming a double-sized ring in the other. This panel is adapted from

Panel B shows the normal and variant chromosomes 9 in some of the cultured cells of the proband; these cells comprised approximately 6% of 70 cells that were examined: panels (i) one normal chromosome 9 and one ring 9, (ii) one normal chromosome 9 and one broken ring 9, (iii) one normal chromosome 9 and a loss of the ring 9, and (iv) one normal chromosome 9 and one "double ring 9".

Based on the above karyotype data, the parents received genetic counseling. As neither of them carried the ring chromosome 9, it was considered extremely unlikely that the rearrangement would recur in future pregnancies. The couple already had three children, and as they were healthy, it was not considered necessary for them to be karyotyped. With regard to their baby, the parents were informed that previously reported cases with this very rare chromosome constitution presented with moderate to severe learning problems in later life, but with a variable degree of severity.

An EDTA blood sample from the child was requested for molecular karyotyping in order to determine the extent of the loss of chromosome 9 material and provide more informed counseling for the parents. DNA was extracted from the blood sample and genome-wide copy number analysis was determined using an Affymetrix[R] Cytogenetics Whole-Genome 2.7M array, according to the manufacturer's instructions. Regions of copy number change were calculated using the Affymetrix Chromosome Analysis Suite software (ChAS) v.1.0.1 and interpreted with the aid of the UCSC genome browser (; Human Mar. 2006 (hg18) assembly).

The array analysis confirmed the initial cytogenetic findings and refined the breakpoints in both the "p" and "q" arms of the ring chromosome (Figure 3). The "p" arm had a 6.7Mb terminal deletion encompassing the interval 199,112bp-6,926,078bp at chromosome regions 9p24.3 to 9p24.1. The "q" arm had a terminal deletion of 1.3Mb encompassing the interval 138,842,015bp-140,171,337bp at chromosome region 9q34.3 (Figure 3); it is likely that the deletions extended to the extreme telomeric end of both arms.



Cytogenetic analysis is a standard test for a referral of ambiguous genitalia. In most cases where there is cytogenetic involvement, it usually involves the rearrangement or loss of a sex chromosome (X or Y). The case reported here represents a much rarer example of an autosome (non-sex chromosome) rearrangement interfering with normal sexual development. To aid genetic counseling, the first step involved a determination of any chromosomal rearrangements in the proband using conventional karyotyping, together with parental analysis to determine the inheritance characteristics of the rearranged chromosome. If one of the parents had a copy of the rearranged chromosome and was phenotypically normal, then it would be less likely that the ring chromosome would be the cause of the physical abnormalities found in the baby. Once it was established that this was a de novo rearrangement, it was important to confirm that it was the only cell line present in the baby. The presence of a normal cell line with no ring chromosome 9, for example, could improve the child's prognosis. The analysis of 70 metaphase cells ruled out significant mosaicism, at least in peripheral blood. A few anomalous cells were found, which might reflect a culturing artifact, or might be due to dynamic mosaicism (3). Dynamic mosaicism occurs as a consequence of sister chromatid exchange (SCE) of the ring chromosome during replication, resulting in several unbalanced chromosome outcomes. The continual production of these abnormal cell lines has the effect of growth retardation in carriers of ring chromosomes.

Subsequent microarray analysis linked our case to previously reported cases in the DECIPHER database (ref 4; These other cases had similar deletions in either the p or q arms, and so were monosomic for only one end of chromosome 9, in contrast to our patient who was monosomic for both ends of chromosome 9. Our data, together with a literature review of terminal deletions for chromosome 9 (Table 1), shows that sex reversal and ambiguous genitalia are associated with deletions of 9p, while deletions in both the p and q arms can lead to cardiac abnormalities.


Routine cytogenetic analysis should be one of the initial tests carried out for referrals of ambiguous genitalia, as an underlying cytogenetic abnormality may be the cause. This case highlights the rare event of an autosomal rearrangement interfering with normal sexual differentiation. Molecular karyotype analysis offered a much higher level of resolution than traditional cytogenetic analysis. It provides more accurate breakpoint data and identifies the extent of deletion/monosomy of a ring chromosome. This in turn helps in the genetic counseling of a family, as more accurate predictions of phenotype can be made based on gene loss.


We acknowledge the technical assistance of Shalinder Singh, Michel Qorri and Amel Al-Murrani for conventional and molecular karyotyping data.


(1.) Jacobs PA, Prackiewicz A, Law P, Hilditch CJ, Morton NE. The effect of structural aberrations of the chromosomes on reproductive fitness in man. II. Results. Clin Genet 1975; 8: 169-78.

(2.) Schinzel A. Catalogue of Unbalanced Chromosome Aberrations in Man, 2nd edition. Walter de Gruyter, Berlin, 2001.

(3.) Gardner RJM, Sutherland GR. Chromosome Abnormalities and Genetic Counseling, 3rd edition. Oxford University Press, New York, 2004.

(4.) Firth HV, Richards SM, Bevan AP, Clayton S, Corpas M, Rajan D, et al. DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources. Am J Hum Genet 2009; 84: 524-33.

(5.) Purandare SM, Lee J, Hassed S, Steele MI, Blackett PR, Mulvihill JJ, et al. Ring chromosome 9 [r(9)(p24q34)]: a report of two cases. Am J Med Genet A 2005; 138A: 22935.

(6.) Lanzi G, Fazzi E, Veggiotti P, Pagliano E, Gariglio M, Bonaglia C, et al. Ring chromosome 9: an atypical case. Brain Dev 1996; 18: 216-9.

Author information

Roberto L.P. Mazzaschi BSc(Hons), Medical Laboratory Scientist [1]

Donald R. Love PhD, FRCPath, FFSc(RCPA), Director [1]

Alice George BSc(Hons) GradDipQS, Technical Head [1]

Salim Aftimos MD, Pediatrician [2]

[1] Diagnostic Genetics, LabPlus and [2] Northern Regional Genetic Service, Auckland City Hospital, Auckland, New Zealand

Author contributions

Roberto Mazzaschi contributed significantly to the analytical work and substantially drafted the article. Donald Love and Alice George substantively wrote parts of the article for critical content. Salim Aftimos conceived the study. The authors declare no conflicts of interest.

Corresponding author

Alice George, Diagnostic Genetics, LabPlus, Auckland City Hospital, PO Box 110031, Auckland 1148, New Zealand. Email:
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Author:Mazzaschi, Roberto L.P.; Love, Donald R.; George, Alice; Aftimos, Salim
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Case study
Date:Aug 1, 2011
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