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Absence of subtelomeric rearrangements in selected patients with mental retardation as assessed by multiprobe T FISH.
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PMID:  23259705     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
ABSTRACT: BACKGROUND: Mental retardation (MR) is a heterogeneous condition that affects 2-3% of the general population and is a public health problem in developing countries. Chromosomal abnormalities are an important cause of MR and subtelomeric rearrangements (STR) have been reported in 4-35% of individuals with idiopathic MR or an unexplained developmental delay, depending on the screening tests and patient selection criteria used. Clinical checklists such as that suggested by de Vries et al. have been used to improve the predictive value of subtelomeric screening. FINDINGS: Fifteen patients (1--20 years old; five females and ten males) with moderate to severe MR from a genetics outpatient clinic of the Gaffree and Guinle Teaching Hospital (HUGG) of the Federal University of Rio de Janeiro State (UNIRIO) were screened with Multiprobe T FISH after normal high resolution karyotyping. No subtelomeric rearrangements were detected even though the clinical score of the patients ranged from four to seven. CONCLUSION: In developing countries, FISH-based techniques such as Multiprobe T FISH are still expensive. Although Multiprobe T FISH is a good tool for detecting STR, in this study it did not detect STR in patients with unexplained MR/developmental delay even though these patients had a marked chromosomal imbalance. Our findings also show that clinical scores are not reliable predictors of STR.
Authors:
Suely Rodrigues Dos Santos; Dértia Villalba Freire-Maia
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-21
Journal Detail:
Title:  Journal of negative results in biomedicine     Volume:  11     ISSN:  1477-5751     ISO Abbreviation:  J Negat Results Biomed     Publication Date:  2012 Dec 
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Created Date:  2012-12-24     Completed Date:  -     Revised Date:  -    
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Nlm Unique ID:  101152210     Medline TA:  J Negat Results Biomed     Country:  -    
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Journal ID (nlm-ta): J Negat Results Biomed
Journal ID (iso-abbrev): J Negat Results Biomed
ISSN: 1477-5751
Publisher: BioMed Central
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Copyright ©2012 Santos and Freire-Maia; licensee BioMed Central Ltd.
open-access:
Received Day: 12 Month: 5 Year: 2012
Accepted Day: 18 Month: 12 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 21 Month: 12 Year: 2012
Volume: 11First Page: 16 Last Page: 16
PubMed Id: 23259705
ID: 3546875
Publisher Id: 1477-5751-11-16
DOI: 10.1186/1477-5751-11-16

Absence of subtelomeric rearrangements in selected patients with mental retardation as assessed by multiprobe T FISH
Suely Rodrigues dos Santos1 Email: surodosan@yahoo.com.br
Dértia Villalba Freire-Maia2 Email: dfm@osite.com.br
1Department of Genetics and Molecular Biology, Federal University of Rio de Janeiro State (DGBM-UNIRIO), Rio de Janeiro, RJ, Brazil
2Department of Morphology and Genetics, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo, SP, Brazil

Background

Chromosomal abnormalities are an important cause of mental retardation (MR) [1]. Most of these abnormalities can be detected by G-banding karyotype analysis, although subtle structural variations are not easily assessed. Most deletions are located near telomeric regions, a common feature in MR patients [2]. Unfortunately, in most patients (60%) the etiology of MR remains unclear [3], despite the use of good quality metaphase preparations in high resolution assays [4].

In subtelomeric regions, the distal segment is shared by many chromosomes while the proximal is larger and shared by a smaller subset of chromosomes [5]. Sequence similarity between non-homologous chromosomes may cause ectopic recombination during meiosis and lead to subtelomeric rearrangements (STRs) [6], with most translocations occurring in these regions [5]. The frequency of STRs in individuals with MR is 4-35% [7], depending on the assays and patient selection criteria used.

Multiprobe T FISH (MT-FISH) [8] is a specific method for detecting STRs in patients with MR who have a normal karyotype; this technique is widely used in this field of investigation and is available commercially. The wide range in the frequency of STR reflects variation in individual responses in this assay, as well as the availability of adequate laboratory infrastructure [9]. These factors must be considered when choosing the appropriate method in studies of STRs.

The primary aim of this study was to assess the usefulness of MT-FISH as a routine diagnostic test for detecting STRs in children and adolescents with early developmental delay, before the onset of MR. A secondary aim was to improve the predictive value of subtelomeric screening in patients selected using the protocol of de Vries et al. [10]. These individuals had evidence strongly suggestive of a chromosomal imbalance, despite a family history of MR.


Materials and methods
Patient selection

Patients were recruited from the genetics outpatient clinic of HUGG-UNIRIO. The cohort consisted of 15 patients (five females and ten males; age range: 1–20 years old) with learning disabilities and/or MR who presented developmental delay. These subjects had a variable family history of learning disabilities, developmental delay or MR and a normal karyotype, as assessed with a high resolution assay. Dysmorphic features, congenital defects or malformations were recorded systematically during clinical examination. An unknown cause of learning disabilities, developmental delay or MR, or uncommon features in known syndromes, were considered as inclusion criteria. Two sets of twins with the same clinical presentation and two second-grade male cousins without similar clinical features were included in the sample. Tests for fragile X syndrome were done in all males and, to exclude other possible causes of MR, the patients were screened for inborn errors of metabolism and underwent serology tests for toxoplasmosis, rubella, cytomegalovirus, herpes virus and syphilis [11]. Subsequently, the clinical criteria of de Vries et al. [10] were applied and a score of 3 was chosen as the minimum for selecting patients for MT-FISH. All parents/guardians of the patients signed an informed consent form prior to participation in the study. Ethical approval was obtained from the appropriate hospital committees. (HUGG – CEP:22/2003; HSP/UNIFESP CEP: 1070/08).

Cytogenetic technique and molecular cytogenetic analysis
Cytogenetic analysis

Cytogenetic analyses done in the Cytogenetic Laboratory at UNIRIO were used to exclude known chromosomal abnormalities. Lymphocyte cultures were established and harvested using standard protocols for high resolution assays. The quality of the metaphase preparations for karyotype analysis and FISH was enhanced by using Cytoclear® (Genial Genetics Upton, Wirral, UK), according to the manufacturer’s protocol.

Molecular cytogenetics

STRs were detected using Chromoprobe Multiprobe T System® kits (Cytocell Inc., Banbury, UK) with slight modification to the protocol. Hybridization, washing and staining were done according to standard protocols. The test was repeated if more than two probes could not be scored in the multiprobe device. If one or two probes could not be scored then a specific telomeric probe was used to assess the abnormal result. If an alteration was confirmed, FISH was done in the parents to assess whether the abnormality was inherited. Photographic documentation was used only in abnormal cases.


Results

Table 1 shows the clinical and laboratory findings for the 15 patients. Ten patients had a familial history of MR, eight had an abnormal electroencephalogram, seven had central nervous system abnormalities detected by tomography or magnetic resonance, seven had mild malformations or birth defects, five had abnormal vision and one had dysacusis.

Normal subtelomeric FISH results were observed in 14 cases. A 13q deletion was suspected in one proband of one set of twins (Figure 1), but a specific telomeric probe did not confirm this (Figure 2). The individual concerned was an eight-year-old boy with severe developmental delay who was a compulsive crier when younger and had mild dysmorphic features. He was the first twin born to a young, healthy, non-consanguineous couple with a family history of MR. His brother had similar clinical features but dissimilar results. FISH analysis of their parents revealed no abnormalities. MT-FISH did not detect STRs in any of the patients.


Discussion

MT-FISH is a precise tool for detecting STRs, but its high cost means that in many developing countries it is first necessary to exclude other more common conditions before choosing this technique. For this reason, we have developed a clinical protocol for investigating the causes of MR [12,13] that can be applied to children suspected of having MR before opting for expensive modern techniques.

Our patients were selected from a large group in which developmental delay was the principal diagnosis and was associated with dysmorphic features and/or malformations. This relationship was initially established in a large screening study in which STRs were found to contribute significantly to unexplained developmental delay and MR with or without a family history of developmental delay or MR [14,15]. In the last decade, several studies using unselected and selected patients have shown that STRs are a prominent cause of MR, especially in idiopathic forms or unexplained developmental delay. Yu et al. [16] reported that the frequency of truly cryptic subtelomeric abnormalities in selected patients was 2.6%. In this study, we examined whether MT-FISH could be used as a routine diagnostic test before the onset of MR. However, this question could not be answered because no STRs were detected in our patients.

Scoring based on clinical criteria has been used in many studies [17-22] to increase the sensitivity of STR detection when MT-FISH is used. De Vries et al. [10] re-examined 29 patients with STRs with regard to their shared characteristics and compared the findings with an MR group without STRs (110 patients). Our group of subjects contained five patients with a low birth weight and nine with a family history of MR. A genetic cause is presumed to underlie half of the cases of undiagnosed patients with idiopathic forms of MR and in many cases there is a family history of MR [12]. Riegel et al. [14] stated that family history was an important selection criterion, especially when MR was associated with dysmorphic features and/or major malformations and growth retardation. As shown here, the number of patients with a low body weight, malformation and a family history was proportionally similar to that reported by de Vries et al. [10]. While the presence of these characteristics and the moderate to severe degree of MR should have increased the likelihood of identifying an STR [10], no STRs were in fact detected. The clinical scores of our patients ranged from four to seven yet no STRs were detected, even though the patients had been carefully selected and were suspected of having a chromosomal anomaly. A similar conclusion was reached by van Karnebeek et al. [23].

Joyce et al. [6] reported one case of STR using MT-FISH that, after a detailed karyotypic review, was found to have semi-cryptic structural anomalies involving a chromosomal imbalance. In the same study, two STRs were found in normal individuals, which suggested polymorphism in subtelomeric regions [24].

A 13q deletion with mosaicism was detected in one of our patients; however, there was no phenotypic correlation with the clinical manifestations [25] and FISH with a specific subtelomeric probe was negative. This case indicates the need to establish a clinical correlation with the cytogenetic findings when an STR is detected, especially if the parents or a specific probe are unavailable.

A non-subtelomeric rearrangement could also explain our negative MT-FISH results. In this case, other techniques such as array comparative genomic hybridization may be useful. Based on our findings, we cannot exclude unknown monogenic disease or multi-factorial conditions as at least part of the etiology of MR and developmental delay in these patients.


Conclusion

In developing countries, there is a need for guidelines for investigating idiopathic MR or unexplained developmental delay in order to establish the best method for a given investigation prior to screening with advanced techniques. MT-FISH is a good tool for investigating the etiology of MR or unexplained developmental delay. However, the cost of this technique in developing countries and the low frequency of STRs mean that the use of MT-FISH may not always be necessary. Finally, clinical criteria were not useful in predicting STRs in this study.


Competing interests

The authors declare that they have no competing interests with the publication of this work.


Authors’ contributions

All authors read and approved the final manuscript.

Suely Rodrigues dos Santos was responsible for designing and doing the experiments and Dértia Villalba Freire-Maia provided supervision and general guidance throughout the study. Both authors were responsible for analyzing and interpreting the results and writing the manuscript.


Acknowledgements

The authors thank Mariluce Riegel for doing the experiments with the specific telomeric probe, Márcia Pimentel for the molecular evaluation of fragile X chromosome and Maria Lúcia C. Oliveira (LABEIM) for assessing the inborn errors of metabolism. Ivonne Banhos provided partial financial support for this work.


References
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Joyce CA,Dennis NR,Cooper S,Browne CE,Subtelomeric rearrangements: results from a study of selected and unselected probands with idiopathic mental retardation and control individuals by using high-resolution G-banding and FISHHum GenetYear: 20011094405110.1007/s00439010058811702226
Knight S,Flint J,Perfect endings: a review of subtelomeric probes and their use in clinical diagnosisJ Med GenetYear: 200037401910.1136/jmg.37.6.40110851249
Mewborn SK,Martin CL,Ledbetter DH,The dynamic nature and evolutionary history of subtelomeric and pericentromeric regionsCytogenet Genome ResYear: 200510822510.1159/00008079815545712
Velagaleti GVN,Robinson SS,Rouse BM,Tonk VS,Lockhart LH,Subtelomeric rearrangements in idiopathic mental retardationIndian J PediatrYear: 2005726798510.1007/BF0272407716131774
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Erjavec-Skerget A,Stangler-Herodez S,Zagorac A,Zagradisnik B,Koklj-Vokac N,Subtelomeric chromosome rearrangements in children with idiopathic mental retardation: applicability of three molecular-cytogenetic methodsCroat Med JYear: 2006478415017167856
DeVries BBA,White SM,Knight SJL,Regan R,Homfray T,Young ID,Super M,McKeown C,Splitt M,Quarrell OW,Trainer AH,Niermeijer MF,Malcolm S,Flint J,Hurst JA,Winter RM,Clinical studies on submicroscopic subtelomeric rearragements: a checklistJ Med GenetYear: 2001381455010.1136/jmg.38.3.14511238680
Curry C,Stevenson RE,Aughton D,Byrne J,Carey JC,Cassidy S,Cunniff C,Graham Jr JM,Jones MC,Kaback MM,Moeschler J,Schaefer B,Schwartz S,Tarleton J,Opitz J,Evaluation of mental retardation: recommendations of a consensus conferenceAm J Med GenetYear: 1997724687710.1002/(SICI)1096-8628(19971112)72:4<468::AID-AJMG18>3.0.CO;2-P9375733
Moeschler JB,Shevell M,and the Commitee on GeneticsClinical genetic evaluation of the child with mental retardation or devepmental delaysPediatricsYear: 200611762304231610.1542/peds.2006-100616740881
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Figures

[Figure ID: F1]
Figure 1 

Deletion suspected by MT-FISH.



[Figure ID: F2]
Figure 2 

No deletion with 13q subtelomeric probe.



Tables
[TableWrap ID: T1] Table 1 

Clinical data, complementary exams and familial history of patients


Patients ID MSA COA JOS CCBN MCSF AJSF YPM JPM RBB RoBB YMB WMC JCTFS LBP LAS
File number
622076
537048
486383
568752
616423
561932
537160
537162
569901
569575
565119
379028
491362
577557
578487
Birthday
10/8/1985
19/9/1988
10/9/1992
19/7/1984
26/9/2004
28/1/2002
20/4/1995
20/4/1995
2/9/1997
2/9/1997
28/4/1992
24/6/1989
2/9/1995
13/10/1998
21/9/2001
Gender
F
F
F
F
F
M
M
M
M
M
M
M
M
M
M
BW (g)
2360
3100
2850
3350
2580
2625
2750
3000
2230
3050
3340
3630
2010
1235
2155
BL (cm)
45
45
48
50
48
47
-
-
-
-
48
48
43
39
-
BHC (cm)
31
-
-
-
32
33
-
-
-
-
-
-
-
31
-
Age (years)
20
17
13
20
115/12
4
10
10
82/12
82/12
13
15
9
6
4
W (kg)
49.5
43.9
67
67.5
29.8
12.5
41.5
39.2
27.5
28
34.4
63.5
23
17
11.3
H (cm)
142
134
156
150
134.5
97.5
137.5
134.5
125
128
137.5
160
121.5
111
100
HC (cm)
50.5
52
52.5
53
51.5
44.5
54
53
51
51
52
58
51
49.5
48
MF
Y
N
N
Y
N
Y
N
N
Y
Y
N
N
N
Y
Y
DD
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Dysmorphism
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
CS
N
N
N
N
N
N
N
N
N
N
Y
Y
N
N
N
FH
N
N
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
LD/MR
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
Y/Y
X fragile
NEG
-
NEG
NEG
-
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NeuroEv
Y
Y
Y
Y
-
Y
Y
Y
Y
Y
-
Y
Y
Y
Y
CCT / MR
ANL
NL
NL
NL
NL
NL
NL
NL
ANL
ANL
ANL
ANL
ANL
NL
ANL
EEG
ANL
-
NL
-
-
ANL
NL
NL
ANL
ANL
ANL
ANL
ANL
ANL
NL
TORCHS
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
ANL
NEG
NEG
IEM
NEG
NEG
I
NEG
NEG
NEG
I
I
I
I
NEG
NEG
NEG
NEG
I
BERA
NL
NL
NL
NL
NL
-
NL
NL
-
-
NL
NL
ANL
NL
NL
Ophtalmo Ev
NL
NL
NL
NL
NL
NL
ANL
ANL
NL
NL
NL
ANL
ANL
NL
ANL
DeVries 8 5 4 5 7 8 4 5 5 7 6 5 8 8 7

Birth Weight (BW); Birth Length (BL); Birth Head Circumference (BHC); Weight (W); Height (H); Head Circumference (HC); Malformation (MF); Developmental Delay (DD); Consanguinity; (CS) Familial History (FH); Learning Disabilities (LD)/ Mental Retardation (MR); Neurologic Evaluation (NeuroEv); Cranio Computerized Tomography (CCT) or Cranio Magnetic Resonance (CMR); Electroencephalography (EEG); Toxoplasmosis, rubella, citomegalovirus, herpes virus and syphilis sorology (TORCHS); Inborn Erros of Metabolism (IEM); otoacustic potencials evoked evaluation (BERA); Ophtalmo Evaluation (Ophtalmo Ev).

Female (F); Male (M); Yes (Y); No (N); Negative (NEG); Inconclusive (I); Normal (NL); Abnormal (ANL) clinical score (DeVries).



Article Categories:
  • Brief Report

Keywords: Developmental delay, Mental retardation, Subtelomeric rearrangements.

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