Characterization of a ring chromossome 22 by molecular cytogenetics
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CASE REPORT
Characterization of a ring chromossome 22 by molecular cytogenetics Caracterização de um cromossomo 22 em anel por citogenética molecular Cleide Largman Borovik1, Roberto Muller2, Ana Lucia Demarchi3, Abram Topczewski4, Luci Black Tabacow Hidal5, Érica Santos6, Veruska Regina Gava Addesso7, Nydia Strachman Bacal8, Marcelo Henrique Wood Faulhaber9, Sulim Abramovici10
ABSTRACT Objectives: To characterize a ring chromosome 22 by means of molecular cytogenetics in a girl with retarded neuropsychomotor development and dysmorphic features. A study carried out using fluorescent in situ hybridization (FISH) with commercially available probes. The ring chromosome 22 was identified as r(22) (p11q13.3) and did not show any significant loss of genetic material. The results confirm the relevance of molecular cytogenetic studies to clarify diagnosis of patients with developmental delay and unspecific dysmorphic features. Keywords: Chromosome aberrations; In situ hybridization, fluorescence; Chromosomes, human, pair 22; Ring chromosomes; Mental retardation
RESUMO Objetivos: Caracterização de um cromossomo 22 em anel por citogenética molecular em uma menina com retardo do desenvolvimento neuropsicomotor e sinais dismórficos. Estudo pela técnica de hibridização in situ fluorescente (FISH) com sondas comerciais. O cromossomo 22 em anel foi identificado como sendo r(22) (p11q13.3), não apresentando perda significativa de material genético. Os resultados encontrados confirmam a importância do estudo citogenético molecular no esclarecimento diagnóstico em casos de retardo do desenvolvimento com sinais dismórficos inespecíficos. Descritores: Aberrações cromossômicas; Fluorescência em hibridização in situ; Cromossomos humanos par 22; Cromossomos em anel; Retardo mental
INTRODUCTION Ring chromosomes arise from terminal breaks on both arms of a chromosome followed by fusion. The amount of genetic material lost in this process depends on the breakpoints and determines the severity of clinical signs. Another mechanism by which ring chromosomes are formed is telomere dysfunction of a chromosome. This mechanism is more often observed in neoplasms and does not lead to any loss of genetic material(1). Rings have been described for all human chromosomes and identified by G banding(2) and are one of the known causes of congenital malformations, even when there is apparently no loss of genetic material. By and large, these abnormalities are believed to be associated with subtelomeric deletions. On the other hand, several authors suggested that autosomal rings might determine a specific phenotype whatever the chromosome involved. Patients with “ring syndrome” usually have severe growth retardation, no malformations at all or only minor anomalies(3-4). The first report of a patient with a ring chromosome 22 was published by Weleber et al., in 1968 (5), and confirmed by Magenis et al., in 1972(6). Although about 60 other patients were described, their phenotypic characteristics did not identify a definite clinical picture. The breakpoints in small rings are difficult to determine, and slight differences could result in phenotypic variations. The patients usually present
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Technical Coordinator of the Genetics Laboratory - Department of Clinical Pathology, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP.
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Speech Therapist, Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP.
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Graduated in Pharmacy and Biochemistry – Department of Clinical Pathology, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Clinical Pathology – Hospital Israelita Albert Einstein, São Paulo, SP.
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Medical coordinator, Department of Clinical Pathology, Hospital Israelita Albert Einstein, São Paulo, SP.
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Department of Pediatrics, Hospital Israelita Albert Einstein, São Paulo, SP. Corresponding author: Cleide Largman Borovik - Hospital Israelita Albert Einstein - Av. Albert Einstein, 627/701 - Ed. Manuel T. Hidal - 1º andar - Setor de Genética Laboratorial - CEP 05651-901 - São Paulo (SP), Brazil - Tel.: (55 11) 3747-2103 - e-mail:
[email protected] Received on June 27, 2003 – Accepted on September 8, 2003
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Borovick CL, Muller R, Demarchi AL, Topczewski A, Hidal LB, Santos E, Addesso V, Bacal NS, Faulhaber MHW, Abramovici S
delayed intrauterine and postnatal development and dysmorphic features including microcephaly, large malformed ears, preauricular pits, hypertelorism, strabismus, epicanthal folds, palpebral ptosis, thick eyebrows and synophrys, wide bridge of the nose and short mandible, malocclusion and malpositioned teeth, high and/or cleft palate, clinodactyly of the fifth toe and partial syndactyly of the second and third toes, hypotonia, variable levels of mental retardation and seizures(2).
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G Figure 1. Patient aged 3y 7m. Observe low height (a, b), hypoplastic nipples (a), facial dysmorphisms (c, d), brachydactyly and clinodactyly of the fifth finger (e, f), and preauricular pit (g).
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We present the case of a girl with retarded neuropsychomotor development and dysmorphic features who presented a ring chromosome 22. FISH technique using commercially available probes did not show any significant loss of genetic material.
CASE REPORT J.S.A., female, born on April 19, 1999, first child of nonconsanguineous parents, was referred to specialized care at the age of 3 years and 7 months (Pediatric Outpatients’ Clinic - Program Einstein in the Community - Paraisópolis) due to retarded neuropsychomotor development. The patient held her head up at 10 months of age, sat down at 2 years, walked at 3 years and spoke the first two-syllable words at 3 years and 7 months. Her mother was 27 years old and had three children from a previous marriage (G4 P4 A0). The mother reported no use of drugs during the uneventful term pregnancy but she reported weak fetal movements as from the third month of gestation. The child was born by vaginal delivery, had no anoxia and weighed 2028 g. No other birth parameters were reported. The newborn remained in the nursery for three days and was discharged from hospital with the mother. On physical examination she presented weight 9530 g, short proportional stature - 86 cm (below the third percentile), large forehead, left preauricular pit, sparse medial eyebrows, telecanthus, bilateral epicanthus, anteverted nostrils, hypoplastic nipples, brachydactyly, clinodactyly of the fifth fingers with hypoplastic medial phalanges (figure 1). The child was submitted to bronchoscopy that revealed signs of moderate laryngomalacia and tracheomalacia. Abdominal and pelvic ultrasounds were normal. CYTOGENETIC ANALYSIS The chromosome analysis was performed using G banding and revealed 46 chromosomes, with a ring chromosome 22 – 46, XX, r(22) (p11q13.3) - (figure 2) in 20 metaphases obtained from peripheral blood lymphocyte culture. Fluorescent in situ hybridization (FISH) was carried at with a commercial probe for DiGeorge syndrome (Vysis Inc.) consisting of a 110kb probe covering segments D22S533, D22S609 and D22S942 in 22q11.2, and a LSI-ARSA (Arylsulfatase A) probe, covering the loci close to telomeres D22S941 and D22S943 in 22q13.3 (figure 3). Two of the 50 metaphases analyzed by FISH did not present a ring chromosome 22 signal. Of a total of 200 interphase nuclei, 20 had signals indicating the presence of a single chromosome 22. We observed two micronuclei with fluorescent signals indicating loss of the ring (figure 4).
Characterization of a ring chromossome 22 by molecular cytogenetics
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Figure 2. G banding on metaphase shows ring chromosome 22; 46,XX,r(22)(p11q13.3)
Figure 4. Interphase nucleus shows signals compatible with presence of only one chromosome 22 and micronucleus with fluorescent signals.
It is noteworthy that sometimes (it is difficult to define how often) the signals visualized on the ring seemed to be stronger than those of the normal chromosome 22 (figure 5).
palpebral ptosis, wide bridge of the nose and short mandible). On the other hand, in addition to severe mental retardation, she presented some clinical features observed in patients with chromosome 22 anomalies, such as preauricular pits, laryngomalacia and tracheomalacia, which are defects of the second brachial arch, associated with chromosome 22 deletions and duplications(2). Other features, such as hypertelorism, epicanthus, brachydactyly and clinodactyly of the fifth finger are very unspecific and present in many chromosome abnormalities. The presence of autosomal rings with no significant loss of genetic material characterizing the “ring syndrome” was described mainly in patients with large A ring chromosomes, such as 2, 4, 7, etc. (3,14-15). It is assumed that ring instability could lead to death of cells with monosomy or trisomy for the abnormal chromosome and growth retardation, but with no other phenotypic consequences. Smaller ring chromosomes or chromosomes with genetic contents that allow cell survival, even when the chromosome is absent or duplicated, could originate monosomic or trisomic cell line with phenotypic consequences. In the present case, ring chromosome instability was demonstrated by the frequency of interphase nuclei (peripheral blood lymphocyte culture) with only one c chromosome 22 (10%) and by micronuclei containing signals compatible with the presence of a ring. On the other hand, the stronger signals visualized on the ring may reflect a duplication undetectable by traditional cytogenetic analysis. Although small, chromosome 22 is rich in genes as compared to other autosomes(16). Thus, small subtelomeric deficiencies, the presence of an even minor monosomic line and duplications resulting from the instability of
DISCUSSION Most patients with a ring chromosome 22 were identified because of mental retardation. There are no consistent clinical signs, but the most frequent findings are compatible with those of partial deletions or duplications of chromosome 22. There are few cases of ring chromosomes 22 characterized by molecular studies so far and in most of them the segment containing the locus ARSA is deleted(7-11). This was not observed in our patient. Wong et al.(12) determined the breakpoint in a patient with a deletion distal to gene ARSA. This patient, described by Flint in 1995(13), presented severe language delay and mental retardation, with no facial dysplasia. Our patient did not present significant abnormalities, such as microcephaly or other dysmorphisms (syndactyly, large malformed ears,
Figure 3. Ideogram of chromosome 22 shows probe location (adapted from www.vysis.com)
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Figure 5. Fluorescent in situ hybridization with DiGeorge/ARSA probe in metaphase (a,b) and interphase nucleus (c). Observe the presence of signals both in normal (22) and ring r(22) chromosome 22.
the ring may explain the clinical signs of this patient. Further studies using subtelomeric probes could better clarify the extent of the lost genetic material.
CONCLUSION The results confirm the relevance of molecular cytogenetics in clarifying the diagnosis of patients with developmental delay and unspecific dysmorphic features. REFERENCES 1. Gisselsson D, Jonson T, Petersen A, Strombeck B, Dal Cin P, Hoglund M et al. Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumours. Proc Natl Acad Sci USA 2001;98:12683-8. 2. Schintzel A. Catalogue of unbalanced chromosome aberrations in man. 2nd ed. Berlin: Walter de Gruyter; 2001. 3. Cote GB, Katsantoni A, Deligeorgis D. The cytogenetic and clinical implications of a ring chromosome 2. Ann Genet 1981;24:231-5. 4. Kosztolanyi G. Does “ring syndrome” exist? An analysis of 207 case reports on patients with a ring autosome. Hum Genet 1987;75:174-9.
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