Mental retardation (MR) is a common disorder found in children with a prevalence of approximately 1%-3% worldwide [1]. The cause of MR is heterogeneous, although majority are unknown. Various types of unbalanced chromosomal rearrangements are the cause of MR in 5%-30% of reported cases [1]. Rearrangements involving subtelomeric regions have been identified as contributing significantly to mental deficiencies, autism, and birth defects, at rates ranging from 1%–30% of cases depending on screening technique and sample selection method [2-10].
Subtelomeres are gene-rich regions. Frequent rearrangement of 1 of these regions is thought to be caused by sequence similarity among nonhomologous chromosomes [11, 12]. Submicroscopic aberrations in 1 of these areas are generally too small to be detected by standard karyotyping using the G-banding technique. The introduction of the fluorescence in situ hybridization (FISH) method has helped to increase the resolution available to study such tiny details and thus increased our ability to detect such cryptic rearrangements; however, FISH is laborious and unsuitable for detecting sequential duplications or small deletions [13]. Whole-genome screening using a microarray technique for detecting any chromosomal rearrangements including subtelomeric aberrations is currently the best available first-tier diagnostic test for individuals with MR and dysmorphism [14, 15].
However, this technique is not available at many diagnostic laboratories because it requires a substantial investment in equipment and resources. Multiplex ligation-dependent probe amplification (MLPA) is another method for subtelomeric screening, and has the advantage of allowing rapid screening of multiple patients in a single assay. This method requires only capillary electrophoresis for fragment analysis. The MLPA technique is reliable and sensitive [16-19]. Additionally, the low cost of MLPA makes it a practical alternative to microarray analysis. Here we report a series of subtelomeric screenings by MLPA in pediatric Thai patients with MR and autism to determine whether implementing MLPA increases the diagnostic yield in our laboratory.
The study was approved by the Faculty of Medicine, Prince of Songkla University, Ethics Committee (approval No. EC 52-138-05-1-3). After written informed consent from their parents or legal guardians of all participants, we collected 129 peripheral blood samples (74 boys, 55 girls, average age = 3.7 years). We diagnosed 114 patients with idiopathic mental retardation (MR) with normal karyotypes detected by a standard G-banding technique. Fifteen patients (12 boys, 3 girls) fulfilled the diagnostic criteria of autistic disorder following DSMIV. All 15 patients with autistic features also had a below average IQ (<70, nonverbal form of the Standford–Binet Intelligence Scale, 5th edition). All had a normal karyotype and no CGG repeat expansion in the FMR1 gene. All autistic girls had no mutation in the coding regions of the MECP2 gene. Common mutation in the ARX genes was excluded in all autistic boys.
DNA was extracted from the peripheral blood samples using the Illustra blood genomic Prep Mini Spin Kit (GE Healthcare, Piscataway, NJ, USA) or standard phenol–chloroform method. The DNA in each sample was quantified and assessed for degradation.
A SALSA MLPA P070-B1 Human Telomere-5 kit (MRC-Holland, Amsterdam, The Netherlands) was used to screen the sample for subtelomeric aberrations. Subtelomeric MLPA probes from a SALSA MLPA P036-E1 Human Telomere-3 kit were used to validate any positive results. The screening was performed using 50 ng of genomic DNA according to the manufacturer’s protocol. A positive control from either a trisomy 21 or a trisomy 13 patient and a negative control sample were included in each MLPA assay. Analysis of the MLPA data was performed as described previously [20].
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PCR reactions were conducted in triplicate using the CFX connect Real-time PCR Detection system (Bio-Rad, Hercules, CA, USA). Fifty nanograms of DNA was used in a 20 μl reaction containing 1× SsoFast EvaGreen Supermix (Bio-Rad), 0.2 μM of each primer for the
We detected 4 types of submicroscopic aberration in samples of 5 patients of 129 from the first screening. Three duplications at chromosome regions 15q11.2, Xp22.33, and 11p15.5, and 1 deletion at 1p36.33, were identified. All patients had been previously diagnosed with idiopathic MR. No abnormality was detected in any individuals diagnosed with autism. Two patients had the same duplication at Xp22.23. All initial results from the first screening were confirmed using different MLPA probes. The deletion of 1p36.33 was confirmed by real-time PCR. The results and the available clinical features of all affected patients are summarized in
Summary of MLPA results and clinical symptoms of all positive cases
Patient | No. Duplication | Verification Method | Sex | Age | Origin | Neurological symptoms | Other anomaly |
---|---|---|---|---|---|---|---|
1 | 15q11.2 | MLPA | Male | 7 | de novo | MR | - |
2 | Xp22.3 | MLPA | Male | 6 | N/A | MR | N/A |
3 | Xp22.3 | MLPA | Male | 6 | N/A | MRseizure | - Bilateral cleft lips |
- Left cleft palate | |||||||
- Thalassemia hemoglobin E trait | |||||||
4 | 11p15.5 | MLPA | Female | 3 | N/A | MR | - Choanal atresia |
- Coloboma of right upper and lower eyelids | |||||||
- Right cleft lip | |||||||
5 | 1p36.33 | Real-time PCR | Female | 1 | N/A | MR | Flat head with frontal bossing |
MR = mental retardation, N/A = information is not available
All 5 families among the affected individuals with submicroscopic aberrations were invited to participate in this research. Unfortunately, 4 of these families declined to participate because of personal beliefs or other reasons, and only 1 family whose child had a duplication of 15q11.2 agreed to further investigation. With consent, the pedigree of the 15q11.2 duplication family is shown in
Because the short arm of chromosome 15 is comprised only of repetitive sequences, the MLPA probe for 15p was relocated to the region of 15q11.2. Although this is not a subtelomeric region, this region is the closest to centromere and to 15p. Aberrations in this region are also known to cause abnormal phenotypes [22-24].
The proband of the family (IV-7) with submicroscopic duplication of 15q11.2 region was a 7-year-old Thai boy with moderate MR. His birth weight was 2700 g, and there was no remarkable history during pregnancy. The physical examination upon arrival found a 20 kg boy who was 117 cm tall. He had normal physical appearance and no dysmorphism was observed. His mother noticed that he had delayed development compared to his siblings since he was 4 months old, but the mother sought no medical opinion. At our first examination, he was able to understand words and short sentences and was now studying at a local kindergarten. His family history showed that he had 2 cousins also diagnosed with MR; the first (IV-9) a 15-year-old boy living in another province, who had been diagnosed with mild mental retardation (IQ = 65) at 8 years old, and the second (IV-10) a 16-year-old boy with MR and with repetitive hand movements; this second cousin had never been to a doctor and could not understand words or sentences. Neither of these cousins had any known dysmorphic features.
MLPA analysis of all available samples from this family (III-3, III-4, III-7, IV-7, IV-10) failed to detect the 15q11.2 duplication except in the proband. His cousin (IV-10) who also had MR did not have this duplication either. Considering the differences between the clinical symptoms of the 2 individuals, this finding was expected.
The subtelomeric regions at both ends of the human chromosome arms are gene-rich. Any unbalanced rearrangement involving genes in these regions can cause mental retardation and an abnormal phenotype. Unfortunately, standard karyotyping, even with high resolution chromosome banding (550 bands), cannot detect such aberrations. In this study, we screened samples from 129 patients with idiopathic MR and autism for any rearrangements with MLPA and detected 5 patients with positive screening (4%), which is within the range reported previously [2-5, 10, 16, 19, 20,25, 26].
The duplication of 15q11.2 detected in our patient was a de novo event. The major anomaly manifested in our affected individual was developmental delay without any dysmorphism. Although there were 2 other affected individuals in the family, the clinical symptoms of each individual seemed to be distinctive, and it is likely that individuals IV-9 and IV-10 are differently affected. The proximal region of chromosome 15q is a region prone to genomic rearrangement. This region contains several low-copy repeats (LCRs) that mediate various duplications and deletions via nonhomologous recombinations [27]. The most well-known syndromes caused by deletions in this region are the Prader–Willi and Angelman syndromes. Mapping our MLPA probes to the region showed that they were centromeric to the
We also had 1 patient with 11p15.5 duplication and 2 patients with Xp22.33 duplication in this study. The 11p15.5 region is known to contain a cluster of imprinted genes that together play an important role in the control of fetal growth. An abnormality in the imprinting mechanism of genes in this region or a uniparental disomy of chromosome 11 can cause either Beckwith–Wiedemann syndrome (BWS) or Silver– Russell syndrome (SRS) [28]. Paternal duplication of the 11p15.5 region has been reported in a few patients with BWS and maternal duplication of the same region can cause SRS [28, 29]. This duplication detected in BWS/SRS can arise as a de novo event or as a result of chromosomal rearrangement from inherited translocation or inversion. In addition to abnormal growth, psychomotor retardation is often detected in this group of patients [28-30]. From the medical records of our patient, in addition to MR, she also had several dysmorphic features (choanal atresia, coloboma of the right upper and lower eyelids, right cleft lip) (
We detected 2 patients with an Xp22.3 duplication in this study, both male with developmental delay (2/129 = 1.6%). Xp22.3 is a gene-rich region, and deletion of any of several genes in this region can cause a variety of abnormal phenotypes such as X-linked ichthyosis with developmental delay, attention deficit hyperactivity disorder, autism, and social communication deficit [31]. However, the clinical significance of the Xp22.3 duplication is still uncertain and has been classified as a normal variant [32, 33], a pathologic change [34-36] or an unclassified rearrangement [37]. The major gene thought to contribute to abnormal phenotypes in this region is the gene for steroid sulfatase (
Terminal deletion of the short arm of chromosome 1 (1p36) is generally difficult to identify by conventional cytogenetics due to the light-staining G-negative bands, but this deletion is thought to be a contiguous gene syndrome [38]. This deletion can be derived from an unbalanced translocation, complex rearrangement or can be merely a simple deletion. A previous study found no correlation between the deletion size and abnormal phenotypes [38]. Common clinical phenotypes of 1p36 deletion include global developmental delay, hypotonia, hearing impairment, heart defects, and distinct dysmorphic features including microcephaly or brachycephaly, midface hypoplasia, deep-set eyes, flat nasal bridge, large anterior fontanelle, and a prominent forehead [38-40]. Because the family of the patient with the 1p36 deletion refused to be part of any further study, we could not perform additional tests that would indicate more conclusively that this deletion derived from any familial chromosomal rearrangement. However, the patient was noted to have frontal bossing with a flat head, which could have been brachycephaly, feature frequently found in patients with 1p36 deletion syndrome. Therefore, the 1p36.33 deletion detected in our study was likely to be pathologic.
Our study shows that implementing subtelomeric MLPA screening in Thai patients with idiopathic mental retardation and autism, who have a normal karyotype, can increase the diagnostic yield. Because some subtelomeric defects may be caused by unbalanced chromosomal rearrangements resulting in a complex submicroscopic aberration, MR and dysmorphism are frequently detected in this group of patients. Currently, a recommended test for MR patients with normal karyotype is microarray analysis, which, although being a useful tool for whole genome screening, is also costly and requires an array facility, which may not be widely available, especially in developing countries. MLPA is an effective alternative way to detect subtelomeric rearrangement of all chromosomes in a single reaction, and is also cheaper and less laborious than FISH. Therefore, we recommend MLPA screening in patients with idiopathic MR with dysmorphism and normal karyotype in diagnostic laboratories where a microarray facility is unavailable.