Genotype-phenotype correlation in 22q11.2 deletion syndrome

  • Elena Michaelovsky1, 2,

    Affiliated with

    • Amos Frisch1, 2, 7Email author,

      Affiliated with

      • Miri Carmel1, 2,

        Affiliated with

        • Miriam Patya1, 2,

          Affiliated with

          • Omer Zarchi1, 3,

            Affiliated with

            • Tamar Green1, 3, 4,

              Affiliated with

              • Lina Basel-Vanagaite1, 2, 5,

                Affiliated with

                • Abraham Weizman1, 2, 6 and

                  Affiliated with

                  • Doron Gothelf1, 3

                    Affiliated with

                    BMC Medical Genetics201213:122

                    DOI: 10.1186/1471-2350-13-122

                    Received: 5 July 2012

                    Accepted: 10 December 2012

                    Published: 17 December 2012

                    Abstract

                    Background

                    The 22q11.2 deletion syndrome (22q11.2DS) is caused by hemizygous microdeletions on chromosome 22q11.2 with highly variable physical and neuropsychiatric manifestations. We explored the genotype-phenotype relationship in a relatively large 22q11.2DS cohort treated and monitored in our clinic using comprehensive clinical evaluation and detailed molecular characterization of the deletion.

                    Methods

                    Molecular analyses in 142 subjects with 22q11.2DS features were performed by FISH and MLPA methods. Participants underwent clinical assessment of physical symptoms and structured psychiatric and cognitive evaluation.

                    Results

                    Deletions were found in 110 individuals including one with an atypical nested distal deletion which was missed by the FISH test. Most subjects (88.2%) carried the 3Mb typically deleted region and 11.8% carried 4 types of deletions differing in size and location. No statistically significant genotype-phenotype correlations were found between deletion type and clinical data although some differences in hypocalcemia and cardiovascular anomalies were noted.

                    Analysis of the patient with the distal nested deletion suggested a redundancy of genes causing the physical and neuropsychiatric phenotype in 22q11.2DS and indicating that the psychiatric and cognitive trajectories may be governed by different genes.

                    Conclusions

                    MLPA is a useful and affordable molecular method combining accurate diagnosis and detailed deletion characterization. Variations in deletion type and clinical manifestations impede the detection of significant differences in samples of moderate size, but analysis of individuals with unique deletions may provide insight into the underlying biological mechanisms.

                    Future genotype-phenotype studies should involve large multicenter collaborations employing uniform clinical standards and high-resolution molecular methods.

                    Keywords

                    Velocardiofacial syndrome (VCFS) Multiplex ligation probe amplification (MLPA) Copy number variation (CNV) Molecular diagnosis Neuropsychiatric disorders

                    Background

                    The 22q11.2 deletion syndrome (22q11.2DS), also known as velocardiofacial syndrome (VCFS) and DiGeorge syndrome, is a genetic disorder caused by hemizygous microdeletions on chromosome 22q11.2, with population prevalence of about 1 to 4,000 births [1]. The 22q11.2DS has multisystem manifestations including conotruncal congenital heart defects (CHD), velopharyngeal anomalies, hypoparathyroidism, T-cell immunodeficiency, craniofacial features, cognitive deficits and high rates of psychiatric morbidity [e.g., schizophrenia, anxiety disorders and attention deficit hyperactivity disorder (ADHD)] [24]. The clinical phenotype is characterized by variable expression and incomplete penetrance.

                    The mechanism mediating 22q11.2 deletions involves non-allelic homologous recombination of low copy repeats (LCR) in 22q11.2. The majority (90%) of the 22q11.2 deletions are of 3Mb (encompassing ~60 genes) occurring between LCR A and LCR D and often referred to as typically deleted region (TDR) while 8% are 1.5Mb in size (~28 genes) (LCR A-B). Some rare atypical deletions of shorter size and in variable locations have also been reported [5]. We have long been interested in 22q11.2DS as a model for studying the genetic contribution to psychiatric disorders and for exploring the correlation between the molecular characteristics of the deletion and the cognitive and psychiatric manifestations in the patients.

                    The classic molecular diagnostic procedure used for detection of deletions and duplications at 22q11.2 is chromosomal analysis coupled with fluorescent in situ hybridization (FISH) using a single fluorescent probe (N25 or TUPLE1) located in the proximal part of the typically deleted region. The FISH technique which is still in routine use in many laboratories is unable to detect deletions that are either proximal or distal to the particular FISH probe used. In recent years several new methods for detecting the 22q11.2 deletion have been developed: comparative genomic hybridization (CGH) [6, 7], multiplex ligation probe amplification (MLPA) [8, 9], multiplex quantitative real-time PCR [10, 11] and high-resolution SNP microarray analysis [10, 12]. The above-mentioned whole-genome methods are still at the research stage and require expensive equipment for performing the assay and analyzing the data. The MLPA method uses multiple probes to achieve a good resolution combined with the practicality and affordability of a commercial kit, which is rapidly and easily performed in any experienced laboratory.

                    We evaluated the utility of MLPA as an alternative to FISH in diagnosing 142 Israeli individuals who had been referred to our clinic with the initial suspicion of 22q11.2DS and also as a tool for characterizing the deletion size and location. The information of the size and location of the deletion in the 22q11.2DS subjects is hereby presented together with their physical and psychiatric clinical features. This may help in the search for a possible genotype-phenotype correlation of different deletion types. The clinical phenotype of individuals with the typically deleted region and other less common deletions are compared by quantitative measures and also by descriptive means. In addition, the physical and longitudinal neuropsychiatric phenotype of an individual with a rare short deletion is described in detail.

                    Methods

                    Subjects

                    The study included 142 individuals referred to the Behavioral Neurogenetics Center (BNC) at Schneider Children’s Medical Center, Petah-Tikva, Israel between January 2001 and December 2009. Mean age 16.1±9.7 years; 78 males (55%) and 64 (45%) females. Sources of referral were: (1) Individuals with FISH confirmed diagnosis of 22q11.2DS referred by clinical genetics departments in Israel; (2) Individuals clinically suspected of the presence of 22q11.2DS, referred by cardiologists, developmental pediatrics, psychiatrists and clinicians at a cleft palate unit.

                    The study protocol was approved by the Rabin Medical Center Review Board, Petah-Tikva, Israel and written informed consent was obtained from the study participants and their parents or guardians.

                    Clinical evaluation

                    Study subjects and their parents underwent a comprehensive medical and developmental intake with a structured clinical checklist composed of the comprehensive multisystem possible clinical symptoms of 22q11.2DS and developmental history [2]. Subjects were referred to further medical evaluations that included cardiac check-up (including electrocardiography and echocardiography), assessment for the presence of typical facial features, evaluation of cleft anomalies (including multi-video fluoroscopy and video endoscopy) and blood calcium levels as previously described [13].

                    Neuropsychiatric evaluation

                    All subjects and their parents were interviewed by skilled clinicians, who were trained until they achieved satisfactory inter-rater reliability scores with the senior investigator (D.G.), using the Hebrew version of the Schedule for Affective Disorders and Schizophrenia for School-Aged Children, Present and Lifetime (K-SADS-PL) [14, 15]. Adults and their parents (when available) were interviewed with the Structured Clinical Interview for Axis I DSM-IV Disorders (SCID) [16]. The ADHD module from the K-SADS was added to the SCID to evaluate the presence of ADHD.

                    Cognitive evaluation was conducted with the age-appropriate versions of the Wechsler Intelligence Scale [17, 18].

                    Laboratory techniques

                    FISH analysis for 22q11.2 deletion was performed according to standard procedures in the cytogenetic laboratory at the Raphael Recanati Genetics Institute, Rabin Medical Center, Israel, using the LSI TUPLE1 (HIRA) or N25 commercial probes (22q11.2) and the LSI ARSA as a sub-telomeric 22q13.3 control probe (Vysis Inc., Downers Grove, IL, USA).

                    DNA extraction

                    DNA from venous blood was extracted using a commercial kit (Promega Corporation, Madison, WI, USA). All DNA samples were quantified by NanoDrop ND 1000 Spectrophotometer (NanoDrop Technologies, City, USA) and stored at −70°C.

                    MLPA analysis

                    MLPA is a PCR- based method designed to detect gene dosage abnormality by relative quantifications. The SALSA MLPA P250-A1 DiGeorge kit (MRC-Holland, Amsterdam, Netherlands) was used to identify copy number variations (CNVs) in the 22q11.2 region (30 probes) and in other chromosomal regions (4q35, 8p23, 9q34, 10p15, 17p13; 18 probes) according to manufacturer's instructions ( http://​www.​mrc-holland.​com). PCR amplification products were separated by capillary electrophoresis using ABI-Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) at the Hy Laboratories Ltd., Rehovot, Israel. The data were analyzed using the Coffalyser VBA analysis software V8 ( http://​www.​mlpa.​com/​coffalyser).

                    Results

                    Molecular analyses

                    Patients were tested for a deletion in 22q11.2 by the FISH technique as part of their routine workup upon admission to the BNC in order to establish the diagnosis of 22q11.2DS. The 22q11.2 deletion was detected in 109 of 140 subjects (77.8%) using either TUPLE1 (HIRA) or N25 commercial probes. The MLPA test was performed on all patients admitted to the BNC with suspected diagnosis of 22q11.2DS, irrespective of their FISH status, as a confirmatory test to FISH and as a tool for determining the deletion location and boundaries. Using the MLPA P250-A1 DiGeorge kit, a 22q11.2 deletion was detected in 110 (77.5%) of the 142 individuals examined. Duplications were not detected in any of the subjects. Additional probes outside 22q11.2 contained in this kit (from chromosomes 4q34, 8p23, 9q34.3, 10p15, 17p13.3 and 22q13) did not reveal additional CNVs (either deletions or duplications), using a criterion of 'more than one consecutive probe showing significant deviation from the normal values'. A detailed graphical representation of the MLPA analysis for 110 individuals with deletions is depicted in Figure  1. Most subjects (97/110; 88.2%) showed a similar pattern of deletion spanning molecular probes from CLTCL1 to LZTR1 representing the 3Mb TDR.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-13-122/MediaObjects/12881_2012_1017_Fig1_HTML.jpg
                    Figure 1

                    MLPA results for 110 individuals with 22q11.2 deletion. Shown below are 30 gene probes within 22q11.2 region (MLPA-P250 kit) which were used for identifying the copy number variation. The copy number is indicated by color: red- one copy (hemizygous deletion); white- two copies.

                    The deletions in 13 individuals (11.8%) differed from TDR and thus were referred to as non-TDR deletions. Four subjects had a 1.5Mb deletion located between CLTCL1 and DGCR8 probes (LCR A-B), four other subjects had a deletion between CLTCL1 and PCQAP (LCR A-C) and a larger deletion extending from CLTCL1 to HIC2 (LCR A-D+) was identified in 4 individuals (Figure  2). One individual (VC901) carried a short (~1Mb) deletion between LCR B and D+.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-13-122/MediaObjects/12881_2012_1017_Fig2_HTML.jpg
                    Figure 2

                    Schematic representation of 22q11.2 deletions detected by MLPA. Genes marked in blue contain MLPA P250-A1 probes. A typically deleted region (TDR) is indicated in red; non-TDR deletions are presented by other colors. FISH probes – N 25 and TUPLE1 (HIRA) are indicated by stars.

                    There was high agreement between the FISH and MLPA results. All patients identified by FISH (N=109) were confirmed by MLPA, but MLPA identified an additional individual with a 22q11.2 deletion (VC901, Figure  1, 2). This deletion could not be identified by FISH since its starting point is distal to the FISH N25 and TUPLE1 probes (Figure  2). All individuals that were found to be non-deleted by MLPA (N=30) were also FISH negative.

                    Since the P-250 MLPA kit lacks sufficient probes in the proximal end of the deletion we used the RT qPCR technique as a supplementary method. We studied the deletion status in the proximal breakpoint using SYBR Green labeled PRODH probe and the VPREB1 probe as a control [11]. It was found that the PRODH gene was hemizygously deleted in most (68/71; 95.8%) subjects tested. Out of 3 deletions that did not contain the PRODH gene two were LCR A-D (~3Mb) and one was LCR A-B (1.5Mb).

                    Genotype-phenotype analysis

                    Tables  1 and 2 summarize the cumulative physical and psychiatric data of the 110 subjects with 22q11.2 deletions grouped according to the type of deletion. There are 97 individuals with TDR and 13 individuals divided into four groups of different types of non-TDR deletions. The small number of individuals in each of the different non-TDR deletions and the inherent variability of the clinical data preclude rigorous statistical analysis. However, it can be seen that there are no large differences between the groups in terms of physical and psychiatric manifestations typical to the 22q11.2DS. It is of note that hypocalcemia was present in 27.7% (28/101) of individuals with 3Mb and larger deletions and in none (0/8) of the individuals with the smaller proximal deletions (1.5 and 2Mb) (Table  1).
                    Table 1

                    Clinical data for 22q11.2DS subjects - Physical features

                    Deletion type

                    Cardiovascular anomalies

                    Palate abnormality

                    Facial dysmorphology

                    Hypocalcemia

                    LCR A-D (TDR)

                    67/97(0.69)

                    80/97 (0.82)

                    85/87 (0.98)

                    27/97 (0.28)

                    LCR A-B (~1.5 Mb)

                    1/4 (0.25)

                    3/4 (0.75)

                    4/4 (1.00)

                    0

                    LCR A-C (~2 Mb)

                    3/4 (0.75)

                    3/4 (0.75)

                    4/4 (1.00)

                    0

                    LCR A-D+

                    4/4 (1.00)

                    4/4 (1.00)

                    4/4 (1.00)

                    1/4 (0.25)

                    LCR B-D+

                    1/1 (1.00)

                    1/1 (1.00)

                    0

                    0

                    Table 2

                    Clinical data for 22q11.2DS subjects - Psychiatric and cognitive features

                    Deletion

                    Age [years] Mean ±SD (N)

                    VIQ

                    PIQ

                    FIQ

                    Psychosis

                    ADHD

                    MDD

                    Anxiety disorders

                    OCD

                    ASDs

                      

                    Mean ±SD (N)

                      

                    SZ

                    SZaff

                    Psychotic dis. NOS

                         

                    LCR A-D 3Mb (TDR)

                    17.39±10.1 (97)

                    77.5±12.6 (81)

                    76.2±13.6 (80)

                    74.2±12.5 (82)

                    7/80 (0.09)

                    1/80 (0.01)

                    3/80 (0.04)

                    36/80 (0.45)

                    8/80 (0.10)

                    32/80 (0.40)

                    8/80 (0.10)

                    3/69 (0.04)

                    LCR A-B (~1.5 Mb)

                    20.50±17.41 (4)

                    81.0+18.0 (3)

                    73.0±14.0 (3)

                    75.0+16.8 (3)

                    0

                    1/4 (0.25)

                    0

                    2/4 (0.50)

                    0

                    1/4 (0.25)

                    0

                    0

                    LCR A-C (~2 Mb)

                    17.00±15.56 (4)

                    79.7+8.5 (3)

                    70.0±2.0 (3)

                    68.8±6.5 (4)

                    0

                    0

                    0

                    3/4 (0.75)

                    0

                    2/4 (50)

                    0

                    0

                    LCR A-D+

                    11.25±2.63 (4)

                    79.3+11.4 (4)

                    75.3±12.3 (4)

                    75.0±10.5 (4)

                    0

                    0

                    0

                    4/4 (1.00)

                    1/4 (0.25)

                    2/4 (0.50)

                    1/4 (0.25)

                    0

                    LCR B-D+

                    20

                    69

                    55

                    59

                    0

                    0

                    1

                    1/1 (1.00)

                    0

                    1/1 (1.00)

                    0

                    0

                    VIQ, PIQ and FIQ – verbal, performance and full IQ, respectively, SZ - Schizophrenia, SZaff - Schizoaffective Disorder, Psychotic dis. (NOS) - Psychotic Disorder Not Otherwise Specified, ADHD - Attention Deficit Hyperactivity Disorder, MDD- Major Depressive Disorder, OCD – Obsessive Compulsive Disorder, ASDs – autism spectrum disorders.

                    Table  3 gives a detailed description of the clinical data of each the 13 non-TDR subjects. Individuals with the 1.5Mb deletion (LCR A-B) exhibited fewer cardiovascular anomalies compared to the other non-TDR subjects (1/4, 25% vs. 8/9, 88.9%; p=0.052).
                    Table 3

                    Clinical data of 22q11.2DS subjects with non-TDR deletion

                    ID

                    Age at diagnosis [years]

                    Deletion

                    Psychiatric diagnoses

                    Cardiovascular anomalies

                    Facial dysmorphology

                    Palate abnormality

                    VC671

                    16

                    LCR A-B (~1.5 Mb)

                    MDD GAD

                    No

                    Eyes, ears, chin, nose

                    Cleft palate

                    VC672

                    46

                    LCR A-B (~1.5 Mb)

                    Schizoaffective disorder

                    No

                    Chin

                    No

                    VC1081

                    7

                    LCR A-B (~1.5 Mb)

                    ADHD

                    No

                    Mouth, ears, chin, nose, face

                    Occult submucous cleft palate

                    VC1161

                    13

                    LCR A-B (~1.5 Mb)

                    ADHD

                    PDA, bicuspid aortic valve

                    Eyes, mouth, ears, chin, nose, face

                    Hypernasal speech

                    VC171

                    40

                    LCR A-C (~2 Mb)

                    MDD, Specific phobia, PTSD

                    PFO, hemitruncus atresia of Rt pulmonary, high origin of Lt pulmonary artery from ascending aorta with big Lt to Rt shunt.

                    Eyes, mouth, ears, nose, face

                    No

                    VC201

                    13

                    LCR A-C (~2 Mb)

                    ADHD, Specific phobia

                    Rt aortic arch with aberrant left subclavian. Vascular ring - diagnosed at birth.

                    Eyes, mouth, ears, nose, face

                    Occult submucous cleft palate

                    VC641

                    7

                    LCR A-C (~2 Mb)

                    ADHD

                    VSD, ASD - spontaneous closure, aplastic Rt carotis and medial Lt upper carotis

                    Ears

                    Occult submucous cleft palate

                    VC871

                    8

                    LCR A-C (~2 Mb)

                    ADHD

                    No

                    Eyes, ears, nose, face

                    Occult submucous cleft palate

                    VC431

                    10

                    LCR A-D+

                    ADHD, OCD

                    Small VSD- spontaneously closed. Aberrant Rt. subclavia from descendent aorta

                    Eyes, mouth, ears, chin, nose, face

                    Occult submucous cleft palate

                    VC511

                    9

                    LCR A-D+

                    ADHD, Specific phobia

                    Two small VSDs

                    Eyes, mouth, ears, chin, nose

                    Occult submucous cleft palate

                    VC741

                    11

                    LCR A-D+

                    ADHD

                    ASD spontaneously closed, abnormal aortic valve.

                    Eyes, mouth, ears, chin, nose, face

                    Occult submucous cleft palate

                    VC1621

                    15

                    LCR A-D+

                    ADHD, MDD

                    Bicuspid aortic valve

                    Eyes, mouth, face

                    Occult submucous cleft palate

                    VC901

                    20

                    LCR B-D+

                    Psychotic dis. (NOS), ADHD, GAD, SA, Specific and Social phobia

                    VSD

                    Eyes, mouth, chin, nose, face

                    Cleft lip and palate

                    ADHD- Attention Deficit Hyperactivity Disorder, GAD- Generalized Anxiety Disorder, MDD- Major Depressive Disorder, PTSD- Posttraumatic Stress Disorder, VSD- Ventricular Septal Defect, ASD- Atrial Septal Defect, PDA- Patent Ductus Arteriosus, PFO- Patent Foramen Ovale SA- Separation Anxiety Disorder, Psychotic dis. (NOS) - Psychotic Disorder Not Otherwise Specified.

                    Case report

                    Patient VC901 carried a distal LCR B-D+ deletion located between ZNF74 and HIC2 probes (Figure  2). We elaborate below on the clinical features of this patient because she carried a rare deletion with only two other similar cases in the literature [19, 20] and may allow studying the impact of the deleted genes in this patient on its particular phenotype. Patient VC901 was referred to our clinic at the age of 10 years as a suspected 22q11.2DS by a trained speech therapist based on her physical and neuropsychiatric profile. FISH analysis using the N25 probe failed to detect a deletion in this individual. Patient VC901 was born with ventricular septal defect (VSD) that was corrected by surgery, cleft lip and palate, retrognathia, hypernasal speech, and short stature (below the third percentile for height, weight and head circumference). The facial features share some similarities with 22q11.2DS (see Figure  3). She has a broad square face, hypotelorism, narrow nasal base and broad nasal bridge, overhanging nasal tip, deviated nasal tip (state post cleft lip repair), short philtrum, narrow mouth, chin dimple and broad neck. Already at the age of 5 years she was diagnosed with dysgraphia and dyscalculia. Results on Wechsler testing at the age of 10 years were: full scale IQ 59 (verbal IQ 69, performance IQ 55). Psychiatric assessment at the age 13 years conducted by K-SADS indicated that Patient VC901 is suffering from severe anxiety disorders including generalized and separation anxiety disorders and specific and social phobias. In addition Patient VC901 was coping with ADHD, predominantly inattentive type. She also exhibited bizarre behaviors which were diagnosed as subthreshold psychotic symptoms. On second evaluation at the age of 20 years Patient VC901 was diagnosed with psychotic disorder not otherwise specified, marked by delusions of reference and recurrent episodes in which she threatened to stab her parents with a knife, with a Positive and Negative Syndrome Scale (PANSS) score of 89. Her cognitive performance showed some improvement compared to the first evaluation with full scale IQ score 67 (verbal IQ 73, performance IQ 65).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-13-122/MediaObjects/12881_2012_1017_Fig3_HTML.jpg
                    Figure 3

                    Facial appearance of Patient VC901 with atypical 22q11.2 deletion. Note the broad square face, hypotelorism, narrow nasal base and broad nasal bridge, overhanging nasal tip, deviated nasal tip (state post cleft lip repair), short philtrum, narrow mouth, chin dimple and broad neck.

                    Individuals with no detected deletion

                    In 32 (22.5%) of 142 subjects referred to the BNC with suspicion of 22q11.2DS no deletions were detected either by MLPA or by FISH. To illustrate the problematic diagnosis of subjects showing some 22q11.2DS-like phenotype but with no apparent deletion we selected 12 individuals with the most pronounced clinical manifestations typical to 22q11.2DS and present them in Table  4.
                    Table 4

                    Clinical data of individuals without 22q11.2 deletion

                    ID

                    Age

                    Gender

                    Palate abnormality

                    Cardiovascular anomalies

                    Facial dysmorphology

                    Hypocalcemia

                    VC411

                    11

                    F

                    Occult submucous cleft palate

                    VSD

                    Eyes, chin, face

                    No

                    VC561

                    19

                    F

                    Occult submucous cleft palate

                    TOF

                    Eyes, mouth, nose, face

                    No

                    VC611

                    19

                    M

                    Occult submucous cleft palate

                    Aortic stenosis

                    Eyes, mouth, face

                    Yes

                    VC631

                    14

                    F

                    Occult submucous cleft palate

                    VSD

                    Eyes, mouth, chin, face

                    No

                    VC761

                    24

                    F

                    Submucous cleft palate

                    PDA

                    Eyes, mouth, nose, face

                    No

                    VC861

                    13

                    M

                    Cleft palate

                    VSD, hypoplastic left ventricle

                    Eyes, nose, face

                    No

                    VC941

                    5

                    M

                    Velopharyngeal insufficiency

                    VSD

                    Mouth

                    No

                    VC951

                    10

                    M

                    Hypernasal speech

                    VSD

                    Nose, face

                    No

                    VC1021

                    23

                    F

                    Occult submucous cleft palate

                    Truncus arteriosus

                    Eyes, mouth, ears, chin

                    Yes

                    VC1031

                    11

                    M

                    Hypernasal speech

                    ASD

                    Mouth, nose, face

                    No

                    VC1201

                    15

                    F

                    Cleft palate

                    Aortic stenosis

                    Face

                    No

                    VC1464

                    7

                    F

                    Palate insufficiency

                    PFO, Pulmonary stenosis

                    Mouth, chin, nose

                    Yes

                    VSD- Ventricular septal defect, ASD- Atrial septal defect, TOF - Tetralogy of Fallot, PDA- Patent ductus arteriosus, PFO- Patent foramen ovale.

                    Interestingly, it appears that the variety and severity of the clinical symptoms in these subjects are very similar to those found in confirmed 22q11.2DS patients.

                    Discussion

                    Analyzing a relatively large 22q11.2DS cohort allowed us to compare the FISH and MLPA methods, perform molecular analysis of the 22q11.2 deletion in our patients and examine the phenotype-genotype relationship.

                    The MLPA technique confirmed all the deletions that were previously detected by FISH in our patients while the FISH assay failed to detect one atypical deletion because it was outside the location of the FISH probes. Thus the MLPA is clearly superior to FISH as a diagnostic tool and a useful method for deletion characterization, essential for genotype-phenotype analysis. CGH and SNP array techniques can also detect atypical deletions but their costs are substantially higher. Other studies have recommended using MLPA as a diagnostic tool [8, 21]. However, MLPA is limited in identifying the deletion boundary that is determined by the availability of probes in the deletion breakpoints. The MLPA kit that we used (P250-A1) lacked probes in the candidate PRODH gene located in the proximal breakpoint which was found to be associated with psychotic disorders in 22q11.2DS [22, 23]. To solve this problem we used qPCR technique and found that the PRODH gene was deleted in most (94.4%) of the 22q11.2DS individuals, as was found previously [6, 24]. The MLPA kit released after we performed our study (P324-A2) already contained several PRODH gene probes.

                    Molecular characteristics of the 22q11.2 deletion

                    We identified 5 types of 22q11.2 deletions. According to the results of the specific MLPA kit used, the proximal starting point for all deletions, except one, was located in the CLTCL1 gene probe, near LCR A (Figure  2). The distal breakpoints of the deletions were located in LCRs B, C, D and D+ thus determining the size and location of the deletions. Ninety seven out of 110 individuals (88.2%) carried the typical ~3Mb deletion (LCR A-D) consistent with frequencies found in other studies (77–88.7%) [20, 21, 2427].

                    The largest deletion (LCR A-D+), a variant of the 3Mb deletions, was present in 3.63% of the subjects compared to 4.5-6.5% in previous reports [8, 24]. The prevalence of the proximal deletions of 1.5Mb (LCR A-B) and 2Mb (LCR A-C), each 3.63% in our subjects, varied largely in other studies [20, 21, 2427], probably due to different sample sizes and resolution of the mapping methods used.

                    Genotype-phenotype relationship

                    Studying the genotype-phenotype relationship in 22q11.2DS is a formidable task. The difficulties are obvious: there are up to 200 possible clinical symptoms [4], the clinical variability is large even in individuals with the same type of deletion and the statistical power is low because most individuals harbor the common 3Mb TDR and only a few with other entities. Yet, it can potentially shed light on the role of the genes that contribute to the physical and psychiatric symptoms expressed in this disorder. The quantitative comparison between 97 individuals with TDR and those with four different types of nonTDR deletions along several physical and neuropsychiatric-cognitive parameters did not reveal statistically significant differences. This could be due to methodological limitations such as sample size, and the complexity of the genetic and epigenetic mechanisms governing the phenotype in 22q11.2DS. Our results are supported by most previous studies that could not demonstrate a correlation between the size and the location of the deletion and the clinical features of the subjects [24, 25, 2830]. However, Rauch et al. (2005) demonstrated a correlation between deletion characteristics and phenotypic expression by assessment of individuals with typical and atypical 22q11.2 deletions, showing that atypical CHD and mild dysmorphism are recognizable feature of atypical distal deletions [20]. Recently, a case-report of monozygotic twins differing in their deletion size and clinical expression was published, indicating a possible role of deletion characteristics for the phenotype [31].

                    In accordance with previous studies [32] hypocalcemia was found in 27.7% of patients with large deletions (3Mb, LCR A-D and LCR A-D+) but was absent in those carrying the proximal nested deletions (1.5 and 2Mb). Interestingly, neither was it observed in patients with distal nested deletions [33]. Although the number of cases is small, this observation may suggest that haploinsufficiency of genes both at the proximal and the distal part of 22q11.2 is required in order to cause hypocalcemia.

                    The presented clinical data of each individual carrying a non-TDR deletion are not in themselves informative but may be of help in the effort to build a common data base. More informed and educated conclusions could be made on this intriguing question by applying unified molecular methods for mapping the 22q11.2 deletion (high resolution methods such as CGH and SNP arrays and next generation sequencing) and using agreed clinical parameters and scoring system for characterizing the phenotype.

                    Individuals without 22q11.2 deletion

                    Before the era of molecular diagnosis patients were diagnosed as VCFS (22q11.2DS) solely based on their clinical symptoms. When molecular genetics methods were introduced it turned out that in about 20% of the patients classified as VCFS no deletion could be detected [20, 21] and the question of the etiology of their phenotype is still unknown. Interestingly, when we examined a subgroup of these individuals it appeared that the variety and severity of clinical symptoms in these subjects are very similar to those found in confirmed 22q11.2DS patients. Subjects may have been misdiagnosed because they had a combination of relatively common symptoms, such as cardiac anomalies and neuropsychiatric disorders, that when considered together phenocopy the 22q11.2DS. It is also possible that these patients do carry very small deletions or even point mutations which are below the resolution of the methods used or they have other microdeletion or microduplication syndromes.

                    The case with atypical nested distal deletion

                    Patient VC901 carries a rare, small deletion that does not include the nested proximal 1.5Mb region which harbors several important candidate genes, thought to be sufficient for producing the syndrome [34]. This case portrays the complexity of the molecular mechanisms controlling the 22q11.2DS phenotype, indicating the possibility of redundancy in causative genes. Since the "classical" candidate genes are not deleted in this person other candidate genes residing in the nested distal region may be responsible for the physical features and neuropsychiatric manifestations. For example, of the candidate genes that have been shown to be involved in cardiac malformations (HIRA, UFD1L, TBX1 and CRKL) [3539]CRKL is one that is located in the distal deletion region and has been found to affect cardiac neural crest derivatives in mice [36]. Its loss may be the causative event for the CHD in this case. In a similar manner, the neuropsychiatric phenotype may be affected by the deletion of PIK4CA and SNAP29 from the distal region which have been demonstrated to be associated with schizophrenia [4043], and not by the putative schizophrenia genes PRODH, COMT, DGCR8 and ZDHHC8. Another genetic mechanism that may affect the phenotype involves control elements in the deleted region that act by cis mechanisms on the expression of the non-deleted candidate genes as in the dosage-sensitive interaction between Tbx1and Crkl in mice [44].

                    The longitudinal neuropsychiatric follow-up of this patient has raised the intriguing question of whether multiple genes in the 22q11.2 region are responsible for the emergence of schizophrenia in 22q11.2DS patients. Her psychiatric developmental trajectory is typical of 22q11.2DS namely, anxiety and subthreshold positive psychotic symptoms during childhood evolving to a full psychotic disorder in adulthood. Yet her cognitive developmental trajectory is not typical, as her IQ scores did not decline and even improved a little during young adulthood, whereas in typical 22q11.2DS patients there is a cognitive decline especially of verbal IQ [45, 46]. This might suggest that the neuropsychiatric manifestations in 22q11.2DS are governed by several sets of genetic elements. It is tempting to speculate that genes such as PIK4CA and SNAP29 that are deleted in our patient may be involved in the emergence of psychotic "positive symptoms" while others, such as PRODH and COMT that are not deleted, play a role in the cognitive decline process.

                    Two other 22q11.2DS cases with similar distally nested deletion have been reported but they are not readily comparable to the present case because of the probands' young age. One was a patient with TOF and facial dysmorphology, last assessed at 9 months of age [19], and the other a 6 year-old with typical facial features, significant developmental delay and increased anxiety suggestive of possible early signs of psychiatric illness [20].

                    Conclusions

                    The MLPA method was accurate and efficient in diagnosing 22q11.2DS in our patients and in defining ambiguous cases. In our opinion, for a mid-size laboratory it represents the best choice as an intermediary technique between the classical FISH method and the high throughput methods such as CGH, SNP arrays and deep sequencing.

                    Although the quantitative genotype-phenotype correlations in our cohort were not statistically significant, the clinical data presented may be added to the body of evidence continually accumulating by interested scientists. Individuals with unique deletions may shed light on specific issues as in the case of patient VC901. Her case suggests that there is redundancy of genes causing physical and neuropsychiatric phenotype in 22q11.2DS and that the psychiatric and cognitive trajectories may be governed by different genes.

                    We believe that the study of the genotype-phenotype correlation would be best conducted using a comprehensive approach of creating a multicenter consortium that will establish a large data base of patients to be characterized by uniform clinical standards and by state-of-the art molecular methods.

                    Abbreviations

                    22q11.2DS: 

                    22q11.2 deletion syndrome

                    ADHD: 

                    Attention deficit hyperactivity disorder

                    ASD: 

                    Atrial septal defect

                    ASDs: 

                    Autism spectrum disorders

                    BNC: 

                    Behavioral Neurogenetics Center

                    CGH: 

                    Comparative genomic hybridization

                    CHD: 

                    Congenital heart defects

                    CNVs: 

                    Copy number variations

                    FISH: 

                    Fluorescent in situ hybridization

                    FIQ: 

                    Full IQ

                    GAD: 

                    Generalized anxiety disorder

                    LCR: 

                    Low copy repeats

                    MDD: 

                    Major depressive disorder

                    MLPA: 

                    Multiplex ligation probe amplification

                    OCD: 

                    Obsessive compulsive disorder

                    PANSS: 

                    Positive and negative syndrome scale

                    PDA: 

                    Patent ductus arteriosus

                    PFO: 

                    Patent foramen ovale

                    PIQ: 

                    Performance IQ

                    Psychotic dis. (NOS): 

                    Psychotic disorder not otherwise specified

                    PTSD: 

                    Posttraumatic stress disorder

                    qPCR: 

                    Quantitative polymerase chain reaction

                    SA: 

                    Separation anxiety disorder

                    SNP: 

                    Single nucleotide polymorphism

                    SZ: 

                    Schizophrenia

                    SZaff: 

                    Schizoaffective disorder

                    TDR: 

                    Typically deleted region

                    TOF: 

                    Tetralogy of Fallot

                    VCFS: 

                    Velocardiofacial syndrome

                    VIQ: 

                    Verbal IQ

                    VSD: 

                    Ventricular septal defect

                    Declarations

                    Acknowledgements

                    This project was funded by the March of Dimes grant # 5-FY06-590 (to D.G. and A.F.). The funding body had no role in study design, data collection, analysis and interpretation of data, writing of the manuscript and the decision to submit it.

                    The departments and institutions where the work was done: Felsenstein Medical Research Center, Petah Tikva, Israel and the Behavioral Neurogenetics Center, Sheba Medical Center.

                    Authors’ Affiliations

                    (1)
                    Sackler Faculty of Medicine, Tel Aviv University
                    (2)
                    Felsenstein Medical Research Center
                    (3)
                    The Child Psychiatry Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center
                    (4)
                    Nes-Ziyyona-Beer Yaakov Mental Health Center
                    (5)
                    Rabin Medical Center, Pediatric Genetics Schneider Children’s Medical Center of Israel and Raphael Recanati Genetics Institute
                    (6)
                    Geha Mental-Health Center
                    (7)
                    Felsenstein Medical Research Center (FMRC), Sackler Faculty of Medicine, Tel Aviv University, Rabin Medical Center

                    References

                    1. Scambler PJ: The 22q11 deletion syndromes. Hum Mol Genet 2000,9(16):2421–2426.PubMedView Article
                    2. Shprintzen RJ: Velo-cardio-facial syndrome: a distinctive behavioral phenotype. Ment Retard Dev Disabil Res Rev 2000,6(2):142–147.PubMedView Article
                    3. Bassett AS, Chow EW, AbdelMalik P, Gheorghiu M, Husted J, Weksberg R: The schizophrenia phenotype in 22q11 deletion syndrome. Am J Psychiatry 2003,160(9):1580–1586.PubMedView Article
                    4. Gothelf D, Frisch A, Michaelovsky E, Weizman A, Shprintzen RJ: Velo-Cardio-Facial Syndrome. J Ment Health Res Intellect Disabil 2009,2(2):149–167.PubMedView Article
                    5. Beaujard MP, Chantot S, Dubois M, Keren B, Carpentier W, Mabboux P, Whalen S, Vodovar M, Siffroi JP, Portnoi MF: Atypical deletion of 22q11.2: detection using the FISH TBX1 probe and molecular characterization with high-density SNP arrays. Eur J Med Genet 2009,52(5):321–327.PubMedView Article
                    6. Bittel DC, Yu S, Newkirk H, Kibiryeva N, Holt A 3rd, Butler MG, Cooley LD: Refining the 22q11.2 deletion breakpoints in DiGeorge syndrome by aCGH. Cytogenet Genome Res 2009,124(2):113–120.PubMedView Article
                    7. Urban AE, Korbel JO, Selzer R, Richmond T, Hacker A, Popescu GV, Cubells JF, Green R, Emanuel BS, Gerstein MB, et al.: High-resolution mapping of DNA copy alterations in human chromosome 22 using high-density tiling oligonucleotide arrays. Proc Natl Acad Sci U S A 2006,103(12):4534–4539.PubMedView Article
                    8. Jalali GR, Vorstman JA, Errami A, Vijzelaar R, Biegel J, Shaikh T, Emanuel BS: Detailed analysis of 22q11.2 with a high density MLPA probe set. Hum Mutat 2008,29(3):433–440.PubMedView Article
                    9. Vorstman JA, Jalali GR, Rappaport EF, Hacker AM, Scott C, Emanuel BS: MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q. Hum Mutat 2006,27(8):814–821.PubMedView Article
                    10. Tomita-Mitchell A, Mahnke DK, Larson JM, Ghanta S, Feng Y, Simpson PM, Broeckel U, Duffy K, Tweddell JS, Grossman WJ, et al.: Multiplexed quantitative real-time PCR to detect 22q11.2 deletion in patients with congenital heart disease. Physiol Genomics 2010,42A(1):52–60.PubMedView Article
                    11. Weksberg R, Hughes S, Moldovan L, Bassett AS, Chow EW, Squire JA: A method for accurate detection of genomic microdeletions using real-time quantitative PCR. BMC Genomics 2005, 6:180.PubMedView Article
                    12. Busse T, Graham JM Jr, Feldman G, Perin J, Catherwood A, Knowlton R, Rappaport EF, Emanuel B, Driscoll DA, Saitta SC: High-Resolution genomic arrays identify CNVs that phenocopy the chromosome 22q11.2 deletion syndrome. Hum Mutat 2011,32(1):91–97.PubMedView Article
                    13. Gothelf D, Presburger G, Levy D, Nahmani A, Burg M, Berant M, Blieden LC, Finkelstein Y, Frisch A, Apter A, et al.: Genetic, developmental, and physical factors associated with attention deficit hyperactivity disorder in patients with velocardiofacial syndrome. Am J Med Genet B Neuropsychiatr Genet 2004,126B(1):116–121.PubMedView Article
                    14. Kaufman J, Birmaher B, Brent D, Rao U, Flynn C, Moreci P, Williamson D, Ryan N: Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL): initial reliability and validity data. J Am Acad Child Adolesc Psychiatry 1997,36(7):980–988.PubMedView Article
                    15. Shanee N, Apter A, Weizman A: Psychometric properties of the K-SADS-PL in an Israeli adolescent clinical population. The Israel journal of psychiatry and related sciences 1997,34(3):179–186.PubMed
                    16. First M, Gibbon M, Spitezer R, Williams J: User's Guide for the Structural Clinincal Interview for DSM-IV Axis I Disorders-Research Version. New York: Biometric Research Department, New York State Psychiatric Institute; 1996.
                    17. Wechsler D: Wechsler Intelligence Scale for Children- Third Eddition Mannual. San Antonio, TX: The Psychological Corporation; 1991.
                    18. Wechsler D: Wechsler Adult Intelligence Scale for Children- Third Eddition Mannual. 3rd edition. San Antonio, TX: The Psychological Corporation; 1997.
                    19. Garcia-Minaur S, Fantes J, Murray RS, Porteous ME, Strain L, Burns JE, Stephen J, Warner JP: A novel atypical 22q11.2 distal deletion in father and son. J Med Genet 2002,39(10):E62.PubMedView Article
                    20. Rauch A, Zink S, Zweier C, Thiel CT, Koch A, Rauch R, Lascorz J, Huffmeier U, Weyand M, Singer H, et al.: Systematic assessment of atypical deletions reveals genotype-phenotype correlation in 22q11.2. J Med Genet 2005,42(11):871–876.PubMedView Article
                    21. Stachon AC, Baskin B, Smith AC, Shugar A, Cytrynbaum C, Fishman L, Mendoza-Londono R, Klatt R, Teebi A, Ray PN, et al.: Molecular diagnosis of 22q11.2 deletion and duplication by multiplex ligation dependent probe amplification. Am J Med Genet A 2007,143A(24):2924–2930.PubMedView Article
                    22. Prasad SE, Howley S, Murphy KC: Candidate genes and the behavioral phenotype in 22q11.2 deletion syndrome. Dev Disabil Res Rev 2008,14(1):26–34.PubMedView Article
                    23. Raux G, Bumsel E, Hecketsweiler B, van Amelsvoort T, Zinkstok J, Manouvrier-Hanu S, Fantini C, Breviere GM, Di Rosa G, Pustorino G, et al.: Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome. Hum Mol Genet 2007,16(1):83–91.PubMedView Article
                    24. Weksberg R, Stachon AC, Squire JA, Moldovan L, Bayani J, Meyn S, Chow E, Bassett AS: Molecular characterization of deletion breakpoints in adults with 22q11 deletion syndrome. Hum Genet 2007,120(6):837–845.PubMedView Article
                    25. Carlson C, Sirotkin H, Pandita R, Goldberg R, McKie J, Wadey R, Patanjali SR, Weissman SM, Anyane-Yeboa K, Warburton D, et al.: Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients. Am J Hum Genet 1997,61(3):620–629.PubMedView Article
                    26. Shaikh TH, Kurahashi H, Saitta SC, O'Hare AM, Hu P, Roe BA, Driscoll DA, McDonald-McGinn DM, Zackai EH, Budarf ML, et al.: Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: genomic organization and deletion endpoint analysis. Hum Mol Genet 2000,9(4):489–501.PubMedView Article
                    27. Saitta SC, Harris SE, Gaeth AP, Driscoll DA, McDonald-McGinn DM, Maisenbacher MK, Yersak JM, Chakraborty PK, Hacker AM, Zackai EH, et al.: Aberrant interchromosomal exchanges are the predominant cause of the 22q11.2 deletion. Hum Mol Genet 2004,13(4):417–428.PubMedView Article
                    28. Kerstjens-Frederikse WS, Kurahashi H, Driscoll DA, Budarf ML, Emanuel BS, Beatty B, Scheidl T, Siegel-Bartelt J, Henderson K, Cytrynbaum C, et al.: Microdeletion 22q11.2: clinical data and deletion size. J Med Genet 1999,36(9):721–723.PubMed
                    29. Kurahashi H, Tsuda E, Kohama R, Nakayama T, Masuno M, Imaizumi K, Kamiya T, Sano T, Okada S, Nishisho I: Another critical region for deletion of 22q11: a study of 100 patients. Am J Med Genet 1997,72(2):180–185.PubMedView Article
                    30. Tutulan-Cunita AC, Budisteanu M, Papuc SM, Dupont JM, Blancho D, Lebbar A, Viot G, Lungeanu A, Arghir A: Atypical presentations of 22q11.2 deletion syndrome: explaining the genetic defects and genome architecture. Psychiatry Res 2012,197(3):356–357.PubMedView Article
                    31. Halder A, Jain M, Chaudhary I, Varma B: Chromosome 22q11.2 microdeletion in monozygotic twins with discordant phenotype and deletion size. Mol Cytogenet 2012,5(1):13.PubMedView Article
                    32. Kobrynski LJ, Sullivan KE: Velocardiofacial syndrome, DiGeorge syndrome: the chromosome 22q11.2 deletion syndromes. Lancet 2007,370(9596):1443–1452.PubMedView Article
                    33. Garavelli L, Rosato S, Wischmeijer A, Gelmini C, Esposito A, Mazzanti L, Franchi F, De Crescenzo A, Palumbo O, Carella M, et al.: 22q11.2 Distal Deletion Syndrome: Description of a New Case with Truncus Arteriosus Type 2 and Review. Mol Syndromol 2011,2(1):35–44.PubMedView Article
                    34. Yamagishi C, Hierck BP, Gittenberger-De Groot AC, Yamagishi H, Srivastava D: Functional attenuation of UFD1l, a 22q11.2 deletion syndrome candidate gene, leads to cardiac outflow septation defects in chicken embryos. Pediatr Res 2003,53(4):546–553.PubMedView Article
                    35. Guris DL, Fantes J, Tara D, Druker BJ, Imamoto A: Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome. Nat Genet 2001,27(3):293–298.PubMedView Article
                    36. Farrell MJ, Stadt H, Wallis KT, Scambler P, Hixon RL, Wolfe R, Leatherbury L, Kirby ML: HIRA, a DiGeorge syndrome candidate gene, is required for cardiac outflow tract septation. Circ Res 1999,84(2):127–135.PubMedView Article
                    37. Guo T, McDonald-McGinn D, Blonska A, Shanske A, Bassett AS, Chow E, Bowser M, Sheridan M, Beemer F, Devriendt K, et al.: Genotype and cardiovascular phenotype correlations with TBX1 in 1,022 velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome patients. Hum Mutat 2011,32(11):1278–1289.PubMedView Article
                    38. Jungerius BJ, Hoogendoorn ML, Bakker SC, Van't Slot R, Bardoel AF, Ophoff RA, Wijmenga C, Kahn RS, Sinke RJ: An association screen of myelin-related genes implicates the chromosome 22q11 PIK4CA gene in schizophrenia. Mol Psychiatry 2008,13(11):1060–1068.PubMedView Article
                    39. Vorstman JA, Chow EW, Ophoff RA, van Engeland H, Beemer FA, Kahn RS, Sinke RJ, Bassett AS: Association of the PIK4CA schizophrenia-susceptibility gene in adults with the 22q11.2 deletion syndrome. Am J Med Genet B Neuropsychiatr Genet 2009,150B(3):430–433.PubMedView Article
                    40. Saito T, Guan F, Papolos DF, Rajouria N, Fann CS, Lachman HM: Polymorphism in SNAP29 gene promoter region associated with schizophrenia. Mol Psychiatry 2001,6(2):193–201.PubMedView Article
                    41. Wonodi I, Hong LE, Avila MT, Buchanan RW, Carpenter WT Jr, Stine OC, Mitchell BD, Thaker GK: Association between polymorphism of the SNAP29 gene promoter region and schizophrenia. Schizophr Res 2005,78(2–3):339–341.PubMedView Article
                    42. Guris DL, Duester G, Papaioannou VE, Imamoto A: Dose-dependent interaction of Tbx1 and Crkl and locally aberrant RA signaling in a model of del22q11 syndrome. Dev Cell 2006,10(1):81–92.PubMedView Article
                    43. Gothelf D, Penniman L, Gu E, Eliez S, Reiss AL: Developmental trajectories of brain structure in adolescents with 22q11.2 deletion syndrome: a longitudinal study. Schizophr Res 2007,96(1–3):72–81.PubMedView Article
                    44. Gothelf D, Eliez S, Thompson T, Hinard C, Penniman L, Feinstein C, Kwon H, Jin S, Jo B, Antonarakis SE, et al.: COMT genotype predicts longitudinal cognitive decline and psychosis in 22q11.2 deletion syndrome. Nat Neurosci 2005,8(11):1500–1502.PubMedView Article
                    45. Pre-publication history

                      1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2350/​13/​122/​prepub

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                    This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.