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Indian Pediatr 2016;53: 786-789 |
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Improving the Diagnosis
of Children with 22q11.2 Deletion Syndrome: A Single-center
Experience from Serbia
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*#Goran Cuturilo,
$Danijela Drakulic,
*@Ida
Jovanovic, $Aleksandar
Krstic, *@Milan Djukic,
*‡Dejan
Skoric, #Marija Mijovic,
@Igor Stefanovic,
$Milena Milivojevic and
$Milena Stevanovic
From *Faculty of Medicine, University of Belgrade;
#Department of Medical Genetics, University Children’s Hospital;
$Laboratory for Human Molecular Genetics, Institute of Molecular
Genetics and Genetic Engineering, University of Belgrade; @Department of
Cardiology, University Children’s Hospital; and ‡Department of
Hematology, University Children’s Hospital; Belgrade, Serbia.
Correspondence to: Dr Goran Cuturilo, University
Children’s Hospital, Medical Faculty, University of Belgrade, Tirsova
10, 11000 Belgrade, Serbia.
Email: [email protected]
Received: June 01, 2015;
Initial review: August 07,
2015;
Accepted: June 14, 2016.
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Objective: The incidence of the 22q11.2 microdeletion among
children who have at least two out of five major clinical criteria for
22q11.2 deletion syndrome.
Design: Prospective study.
Setting: University Children’s Hospital in
Belgrade, Serbia between 2005 and 2014.
Participants: 57 patients with clinical
characteristics of 22q11.2 deletion syndrome.
Methods: Standard G-banding cytogenetic analysis
was performed in all children, and the 22q11.2 genomic region was
examined using fluorescence in situ hybridization (FISH). For
patients with no deletion detected by FISH, multiplex ligation-dependent
probe amplification (MLPA) analysis was also done in order to detect
cryptic deletions of this region and to analyze other genomic loci
associated with phenotypes resembling the syndrome. A selected group of
patients diagnosed to have 22q11.2 microdeletion by FISH underwent MLPA
testing in order to characterize the size and position of deletion.
Outcome Measure: The frequency of 22q11.2
microdeletion among children with at least two of
the five major characteristics of 22q11.2 deletion syndrome (heart
malformations, facial dysmorphism, T-cell immunodeficiency, palatal
clefts and hypocalcemia/hypoparathyroidism)
Results: Typical 22q11.2 microdeletion was
detected in 42.1% of patients; heart malformation were identified in all
of them, facial dysmorphism in 79.2%, immunological problems in 63.6%,
hypocalcemia in 62.5% and cleft palate in 8.3%.
Conclusions: A higher detection rate compared to
one-feature criterion is obtained when at least two major features of
22q11.2 deletion syndrome are taking into consideration. The criteria
applied in this study could be considered by centers in low-income
countries.
Keywords: DiGeorge syndrome, fluorescence in situ
hybridization; multiplex ligation-dependent probe amplification.
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22 q11.2 deletion syndrome (22q11.2DS) is the most
common microdeletion syndrome with an estimated incidence of
approximately 1/4000 per live births [1]. More than 180 malformations
are associated with 22q11.2 microdeletion; the most common are cardiac
defects, a characteristic facial appearance, thymic hypoplasia, cleft
palate/velopharyngeal insufficiency (VPI), hypoparathyroidism with
hypocalcaemia, speech and language impairment and developmental delay
[2]. Most patients (87%)
have a deletion of 3 Mb; less frequently (10%) a loss of 1.5 Mb; while a
few patients have unique deletions, translocations or point mutations of
the TBX1 gene [2,3]. Commonly used methods for detection of
22q11.2 microdeletion are FISH and MLPA [1].
Detection rate is an important issue to consider in
terms of achieving a balance between patient coverage and costs.
Previously, different recruitment criteria have been applied providing
22q11.2 microdeletion detection rates ranging from zero to 34.7% [4-15].
We present our 10-year experience with an approach, resulting in high
detection rate, which could be beneficial for centers in low-income
countries.
Methods
Our study included 57 Caucasian children (21 females,
age 1 day-14 yr) (Fig. 1), recruited at the University
Children’s Hospital during the period 2005 - 2014. We wished to assess
the 22q11.2 microdeletion detection rate in a cohort of patients whose
enrollment was based on the presence of at least two out of the five
major characteristics of 22q11.2 deletion syndrome (heart malformations,
facial dysmorphism, T-cell immuno-deficiency, palatal clefts and
hypocalcemia/hypoparathyroidism). Written informed consent was obtained
from the patients’ parents. The Ethical Committee of the University
Children’s Hospital approved the study protocol.
In order to determine presence of the five major
phenotypic features, all patients underwent dysmorphology assessment
provided by a clinical geneticist, as well as further clinical
examinations, including echocardiography, immunophenotyping of
peripheral blood lymphocytes and measurement of serum calcium (if
decreased, additional measurement of parathyroid hormone).
Standard G-banding cytogenetic analysis was performed
on phytohemagglutinin-stimulated peripheral blood lymphocytes according
to routine protocol for karyotyping. Flourescent in situ
hybridization (FISH) analysis on metaphase spreads from cultivated
lymphocytes and multiplex ligation-dependent probe amplification (MLPA)
analysis using Kit P250-A1 DiGeorge (MRC-Holland, The Netherlands) were
carried out as described in Cuturilo, et al. [16]. The applied
high density MLPA kit enables detection of cryptic deletions of the
22q11.2 region and analysis of another five genomic loci associated with
phenotypes resembling 22q11.2DS. All patients enrolled in the study were
screened for hypocalcemia.
The Chi-square test was used to compare differences
in the frequency of occurrence of the typical facial dysmorphism between
patients with and without 22q11.2 microdeletion; a P value <0.05
was considered as statistically significant.
Results
A normal karyotype was detected in 56 patients. In
one case cytogenetic analysis identified a distal 4q deletion. In 27 out
of 33 patients having no deletion detected by FISH, MLPA analysis was
performed (Fig. 1).
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Fig. 1 Flow of patients in the
study.
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Among the 24 patients diagnosed to have 22q11.2
microdeletion by FISH (Table I), 14 patients underwent
MLPA testing in order to characterize the size and position of the
deletion. This analysis revealed 3 Mb deletion in 13 cases, while in one
patient a 1.5Mb deletion was detected.
TABLE I Microdeletion Detection in Children With Combination at least Two Out of Five Major Clinical Criteria
for 22q11.2 Deletion Syndrome (N=24)
Heart Malformation with |
No. |
Facial dysmorpism |
6 |
T-cell immunodeficiency |
2 |
Hypocalcemia |
2 |
Cleft palate |
0 |
Facial dysmorpism and hypocalcemia |
8 |
Hypocalcemia and T-cell immunodeficiency |
1 |
Facial dysmorpism and T-cell immunodeficiency |
1 |
Facial dysmorpism, T-cell immunodeficiency and |
|
hypocalcemia |
2 |
Facial dysmorpism, cleft palate and hypocalcemia |
2 |
The frequency of occurrence of different congenital
heart malformation types are shown in Table II. Dysmorphic
facial features were present in 51 (89.5%) participants. Typical facial
dysmorphism (TFD) (short palpebral fissures, prominent nasal bridge
and/or small mouth [17]) was observed in 20 patients (11 with the
deletion). There was no difference in incidence of TFD between patients
with and without 22q11.2 microdeletion (P=0.53). Non-specific
facial dysmorphism (NSFD) was observed in 31 cases (8 with the
deletion).
TABLE II Type of Congenital Heart Diseases and 22q11.2 Microdeletion
Congenital heart disease |
22q11.2 microdeletion
|
|
Present (no.) |
Absent (no.) |
Tetralogy of Fallot |
6 |
16 |
Pulmonary artery atresia |
4 |
6 |
Common arterial trunk |
5 |
3 |
Interrupted aortic arch |
6 |
1 |
Ventricular septal defect |
2 |
3 |
Double outlet right ventricle |
0 |
2 |
Aorto-pulmonary window |
0 |
1 |
Mitral stenosis |
1 |
0 |
Transposition of great arteries |
0 |
1 |
Decreased T-cell number was detected in seven
patients with the 22q11.2 deletion. The incidence of overt cleft palate
was 3/57 (5.3%), while microdeletion was disclosed only in one case.
Furthermore, another patient with the 22q11.2 microdeletion and nasal
speech was diagnosed with a submucous cleft palate.
Hypocalcemia was detected in 17 (29.8%) subjects
among whom 15 had the microdeletion; hypoparathy-roidism was confirmed
in all but one of the patients with hypocalcemia.
Discussion
Diagnosis of 22q11.2DS is directed towards early
recognition and management, including multidisciplinary follow-up of the
patients. Here we report our ten-year-experience in applying strict
criteria for patient recruitment (presence of at least two out of the
five major clinical characteristics of 22q11.2DS).
Many investigations of the 22q11.2 microdeletion
detection rate in patients with a single phenotypic feature have found a
very low rate, ranging from zero to 17.9% [5,7,9,10,12,15]. In contrast,
other reports have suggested testing for microdeletion 22q11.2 only in
patients with at least two 22q11.2DS features, with detection rates
between 6.2 and 34.7% [4,6-8,10,11,13,14].
Our results provide additional support for using at
least two of the five major clinical characteristics of 22q11.2DS for
patient recruitment in order to achieve a satisfactory detection rate.
Furthermore, the rate we obtained is higher than that of other studies.
A possible explanation is detailed analysis of the phenotype of each
patient by a clinical geneticist, implying that the detection rate for
22q11.2 microdeletion does not depend strictly on the number of
anomalies, but also on a certain level of suspicion arising from the
clinician’s knowledge and experience.
The limitation of our approach is diminished coverage
of patients, primarily those with a less typical 22q11.2DS phenotype.
Furthermore, lack of MLPA testing of all patients diagnosed to have
22q11.2 microdeletion by FISH is another limitation of our study.
In conclusion, analyzing the patients with at least
two major clinical features of 22q11.2DS we obtained a much higher
detection rate for 22q11.2 microdeletion compared to the one-feature
criterion. Furthermore, by applying both FISH and MLPA techniques, we
could detect the typical 22q11.2 microdeletion and cryptic deletions of
the 22q11.2 region. We could also analyze another five genomic loci
associated with phenotypes resembling 22q11.2DS. Overall, this sets the
basis for better care of children with 22q11.2DS in Serbia. Moreover, it
could be interesting for other centers in low-income countries.
Contributors: All authors participated equally in
all aspects of this work.
Funding: This work was supported by the Ministry
of Education, Science and Technological Development, Republic of Serbia
(D.D., A.K., M. Milivojevic and M.S. Grant No. 173051).
Competing interests: None stated.
What is Already Known?
• Different criteria in patient recruitment
for 22q11.2 microdeletion testing have been applied, providing a
22q11.2 microdeletion detection rate of 0 - 34.7%.
What This Study Adds?
• Recruitment based on the presence of at
least two major phenotypic features enables a detection rate as
high as 42.1%.
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