Approach Considerations
Laboratory studies used in the evaluation of 22q11.2DS include the array comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH), TBX1 gene sequencing or TBX1 deletion/duplication analysis, and multiplex ligation-dependent probe amplification (MLPA).
Genetic Studies
Array comparative genomic hybridization (aCGH) is the preferable and most appropriate test for detecting the 22q11.2 deletion. It has the added benefit of detecting large or submicroscopic chromosomal deletions/duplications on all chromosomes in addition to the classic chromosome 22q11.2 deletion. It may also provide refinements of the breakpoints, although the size of the deletion has not yet shown any clinical correlation. [44]
Fluorescent in situ hybridization (FISH) is readily available with chromosome analysis but smaller deletions may not be detectable. If aCGH is unaffordable/unavailable, a FISH study for the 22q11.2 deletion with a karyotype should be requested. The karyotype detects chromosome rearrangements and other chromosomal abnormalities.
Request TBX1 gene sequencing or TBX1 deletion/duplication analysis when aCGH does not show a deletion yet the syndrome is clinically suspected. This should be performed after consultation with a clinical geneticist.
Multiplex ligation-dependent probe amplification (MLPA) appears to be equivalent to the FISH technique [45] and can be used for rapid diagnosis when the syndrome is suspected clinically or for confirmation of the deletion after aCGH analysis. Additionally, an MLPA analysis has been designed to be performed on DNA extracted from dried blood spot samples obtained from Guthrie cards collected via the newborn screening program. [46]
Other means of diagnosis are being evaluated, including a rapid polymerase chain reaction (PCR) assay–based method.
Indications for Deletion Screening
Indications for 22q11.2DS screening depend on the clinical picture. The 22q11.2 deletion occurs in 20-30% of newborns with isolated conotruncal cardiac malformations. Therefore, screening all newborns with conotruncal anomalies for 22q11.2 deletions is well justified. Some other candidates for screening are neonatal hypocalcemia (74%), interrupted aortic arch (50-60%), and velopharyngeal insufficiency (64%). Only about 1% of cases with any cardiac lesion detected later in life and 0-6% of cases of isolated schizophrenia (0-6%) may have 22q11.2DS, thus these facts may warrant an evaluation by a clinical geneticist for advice regarding screening for 22q11.2DS.
The signs and symptoms suggesting 22q11.2DS also depend on the patient’s age at evaluation. Generally, however, 2 or more of the following clinical findings should prompt a laboratory confirmation of the diagnosis:
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Conotruncal heart anomalies
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Palatal defects
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Hypernasal speech
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Nasopharyngeal reflux
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Developmental/learning disabilities
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Behavioral/psychiatric problems
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Immunodeficiency
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Hypocalcemia
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Typical facial features
Laboratory testing
CBC count
A CBC with an elevated mean platelet volume above 10 fL may be a useful screening test involving no extra laboratory work, cost, or patient discomfort. Authors of a retrospective study suggested that the platelet finding may help the clinician to rapidly decide whether to order irradiated blood products to prevent potentially fatal transfusion-associated graft versus host disease in case of severe immune deficiency and may alert clinicians to monitor serum calcium levels closely to prevent hypocalcemic seizures. [47]
Calcium and parathyroid hormone studies
Hypocalcemia may occur in 22q11.2DS (chromosome 22q11.2 deletion syndrome; DiGeorge syndrome [DGS]) secondary to hypoparathyroidism. Measure the ionized serum calcium level to evaluate parathyroid function. If the level is low, obtain simultaneous ionized serum calcium and parathyroid hormone levels. Consult an endocrinologist. Latent or subclinical hypoparathyroidism can be unmasked by performing a diagnostic ethylenediaminetetraacetic acid (EDTA) challenge test. Despite occasional normal calcium and parathyroid hormone levels, the secretory reserve for parathyroid hormone is usually diminished in patients with 22q11.2DS.
Dilated retinal examination
A dilated retinal examination can help to detect familial exudative vitreoretinopathy. [41]
T-Cell Evaluation
Perform an absolute lymphocyte count in the peripheral blood. If lymphopenia is present, consult an immunologist and obtain T- and B-cell counts.
Note that a normal-sized thymus does not necessarily ensure normal T-cell development, and patients with a very small thymus, even in an ectopic location, may have T-cell responses to mitogens that range from below normal to normal. Mitogen responsiveness may be the most important parameter in assessing T-cell function, and peripheral T-cell numbers may not be indicative of T-cell responses.
In other words, although a finding of very low to absent T cells in the peripheral blood suggests severe immunodeficiency, decisions regarding treatment should be based on T-cell proliferative responses to antigens and mitogens, not on the number of T cells.
Newborn screening of severe combined immunodeficiency (SCID), which detects TREC levels in dried blood spots, is likely to identify 22q11.2DS as decreased TREC levels. Newbon screening of SCID may facilitate diagnosis of this condition at an earlier age.
Flow cytometry
Flow cytometry is performed in vitro to estimate the number of T cells in peripheral blood and their proliferative responses to mitogens and antigens. Flow cytometry studies measuring CD45RA+ T and CD45RO+ cells should also be performed, to distinguish patients with complete DiGeorge syndrome from patients with the more common partial DiGeorge syndrome. [48]
Advances in multicolor flow cytometry, noninvasive imaging techniques, and molecular assessments of thymic function have enabled a more comprehensive characterization of human thymic output in clinical settings than in the past. These techniques have been particularly valuable in monitoring reconstitution of T cells after therapeutic thymic grafting for complete 21q11.2 DS
RT PCR assay
T-cell receptor excision circles (TRECs) are small episomal pieces of DNA formed during the rearrangement of T-cell receptor genes of thymocytes undergoing differentiation in the thymus.
The use of real-time, quantitative reverse transcriptase (RT) PCR methods for TREC quantification provides a novel tool for estimating recent thymic function in different clinical situations, including in patients with 22q11.2DS and in persons undergoing thymic transplantation.
In newborn screening, the TREC assay is performed on DNA isolated from the Guthrie card blood spots. Decreased TRECs as a measure of decreased thymopoiesis are seen in infants with congenital T-cell defects, such as SCID, idiopathic T lymphopenia, ataxia telangiectasia, as well as 22q11.2DS. However, very mild form of 22q11.2DS with little thymic hypoplasia may not be detected with this screening measure.
Antibody response
At times, a sudden increase in CD3+/CD4+ T cells is observed in patients with DGS and is associated with a modest mitogen response but no proliferative response to antigens. Response to antigens is the best predictor of the ability of the T cells to protect against infection and is the most clinically relevant of the in vitro tests of T-cell function.
Evaluation of humoral immunity reveals variable immunoglobulin levels and depends on the extent of T-cell deficiency. As would be expected (ie, because normal B-cell development requires normal T-cell function), the B-cell repertoire is normal in patients whose only measurable T-cell defect is a low number. Patients with partial DGS generate good antibody response to protein vaccines, but no data are available on polysaccharide vaccines. Increased prevalence of immunoglobulin A deficiency was observed in 4 of 32 patients with 22q11.2 deletion.
Imaging Studies
Radiography and MRI
Perform chest radiography and other imaging studies based on the cardiologist's recommendations. Chest radiography can reveal a decreased thymic silhouette but is unreliable for thymus assessment. Magnetic resonance imaging (MRI) is slightly better; however, thymic size evaluation is not recommended, because it is a poor predictor of immune function.
Radiography of the head may reveal CNS calcifications, while radiography of the abdomen can depict nephrocalcinosis; however, routine radiography of these structures is unnecessary.
CT scanning
Although computed tomography (CT) scanning of the thorax and angiography in patients with 22q11.2DS may show the following, echocardiography is preferred to CT scanning to avoid radiation:
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Malformations of the heart and great vessels
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Interrupted aortic arch
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Tetralogy of Fallot
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Truncus arteriosus
CT scanning of the neck in patients with 22q11.2DS may show lower carotid artery bifurcations and thyroid abnormalities, including the absence of a lobe, absence of isthmus, and retrocarotid or retroesophageal extension, [49] but usually is unnecessary. MR angiography of the neck is preferable if surgery is planned on the neck.
An absent thymus or one in an aberrant location may be noted on chest radiographs and CT scans. However, despite the emphasis on thymic defects in the literature about DGS, they are clinically significant in less than 5% of cases. Maldescent of the thymus is extremely common. Therefore, CT scanning of the neck and chest is not routinely recommended.
Echocardiography
The range of cardiovascular anomalies in chromosome 22q11.2DS is wide, although conotruncal defects are the most frequent ones. Because slight variations in a defect may dictate which surgical intervention is used, 2-dimensional (2-D) and color Doppler echocardiography are essential to define the anatomy. The thymus may also be visualized in this way. (Although cardiac catheterization may not be needed, it can provide helpful information in some situations.)
Angiography
Either magnetic resonance angiography (MRA) or conventional angiography is necessary to identify abnormalities of the internal carotid arteries before neck surgery is performed.
Prenatal Diagnostic Testing
Prenatal diagnostic testing for chromosome 22q11.2DS can be offered to at-risk couples. [50, 51] Indications for such testing include a previous child or a parent with the syndrome or the in utero detection of a conotruncal cardiac defect. [52] Any of the following are selected based on the clinical situation:
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FastFISH: A rapid FISH method that allows the release of accurate results the same day that amniocentesis is performed [54]
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High-resolution single-nucleotide polymorphism (SNP) genotyping assay [55]
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Chromosomal microarray analysis (CMA): A high-resolution genome scanning procedure for DNA dosage aberrations; it can detect single copy number aberrations
Histologic Findings
In a skin biopsy study, Selim et al concluded that dyskeratotic keratinocytes, satellite cell necrosis, and parakeratotic scale with neutrophils characterize the cutaneous rash seen in patients with a form of complete DGS known as atypical complete DGS. Thus, according to the report, these lesions in patients with DGS should indicate to a pathologist that this relatively rare form of the syndrome may be present. [56] Thymic biopsy findings in DGS are essentially normal except for evidence of hypoplasia.
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Mother and children with 22q11.2 deletion syndrome.
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An African American girl with 22q11.2 deletion syndrome.
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The same child as in the previous image, showing an asymmetrical crying face.
