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2q23.1 Microdeletion Syndrome and Autism Spectrum Disorder via the MBD5 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing and Del/Dup

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
4183 MBD5$640 81479,81479 Add to Order
Pricing Comments

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

One study identified a de novo nonsense variant in MBD5 in 1 of 78 (~1%) individuals with moderate to severe intellectual disability of unknown cause (Bonnet et al. 2013).

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Clinical Features

Autosomal dominant intellectual disability via 2q23.1 microdeletion syndrome is a neurocognitive disorder characterized by moderate to severe intellectual disability and motor delay. 2q23.1 microdeletion syndrome patients often present in infancy with hypotonia and feeding difficulties. During childhood patients may develop seizures, stereotypic hand movements, sleep disturbances, short attention span and autistic-like behaviors (Talkowski et al. 2011). 2q23.1 microdeletion syndrome patients exhibit severe language impairment, often not being able to speak meaningful words. Physical features of patients with 2q23.1 microdeletion syndrome include thick arched eyebrows, broad forehead, downturned mouth and thin upper lip (Hodge et al. 2013). Gross deletions of the 2q23.1 region results in a more severe clinical phenotype than variations that affect only the MBD5 gene, suggesting other genes in the deletion interval contribute to the phenotype. Additional features seen in large 2q23.1 deletion cases include an ataxic gait, self-injurious behaviors, hyperphagia and obesity, pinnae abnormalities, microcephaly and short fifth digit of hands and feet (Talkowski et al. 2011). 2q23.1 microduplications have also been reported in patients with intellectual disability. The most common features of 2q23.1 microduplication syndrome are psychomotor delay, language impairment, infantile hypotonia, behavioral problems and autistic-like symptoms (Mullegama et al. 2013). Shared facial features observed in patients include downslanting palpebral fissures, arched eyebrows, long eyelashes, prominent nose and pinnae abnormalities.

Genetics

2q23.1 microdeletion syndrome is caused by deletions encompassing the MBD5 gene. 2q23.1 microdeletion syndrome is an autosomal dominant disorder and many cases are sporadic, resulting from de novo deletions encompassing the MBD5 gene. Causative nonsense and frameshift mutations in MBD5 have also been reported in patients with intellectual disability (Kleefstra et al. 2012; Bonnet et al. 2013). A number of missense variants in MBD5 have been reported in patients with intellectual disability or autism, but the causal nature of these variants is not yet clear (Wagenstaller et al. 2007; Cukier et al. 2012; Talkowski et al. 2011) MBD5 encodes a protein with a methyl-CpG binding domain that belongs to the same structural family as MeCP2. MBD5 localizes to heterochromatic regions of DNA around centromeres, but has not been shown to bind directly to methylated DNA (Laget et al. 2010). It is proposed that MBD5 directs epigenetic regulation of neuronal gene expression.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the MBD5 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

MBD5 testing should be considered in patients with clinical features of 2q23.1 deletion syndrome. In addition, MBD5 testing should be considered in patients diagnosed with Angelman, Rett, Smith-Magenis or other clinically overlapping syndromes but for which no genetic cause has been identified (Van Bon et al. 2009).

Gene

Official Gene Symbol OMIM ID
MBD5 611472
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Mental Retardation, Autosomal Dominant 1 156200

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autism Spectrum Disorders Sequencing Panel with CNV Detection
Comprehensive Epilepsy and Seizure Sequencing Panel with CNV Detection
Rett Syndrome, Angelman Syndrome and Variant Syndromes Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bon BW Van, Koolen DA, Brueton L, McMullan D, Lichtenbelt KD, Adès LC, Peters G, Gibson K, Novara F, Pramparo T. 2009. The 2q23. 1 microdeletion syndrome: clinical and behavioural phenotype. European journal of human genetics 18: 163–170. PubMed ID: 19809484
  • Bonnet C, Khan A Ali, Bresso E, Vigouroux C, Béri M, Lejczak S, Deemer B, Andrieux J, Philippe C, Moncla A, Giurgea I, Devignes M-D, et al. 2013. Extended spectrum of MBD5 mutations in neurodevelopmental disorders. European Journal of Human Genetics. PubMed ID: 23422940
  • Cukier HN, Lee JM, Ma D, Young JI, Mayo V, Butler BL, Ramsook SS, Rantus JA, Abrams AJ, Whitehead PL, Wright HH, Abramson RK, et al. 2012. The Expanding Role of MBD Genes in Autism: Identification of a MECP2 Duplication and Novel Alterations in MBD5 , MBD6 , and SETDB1: ASD patients with novel variants in MBD genes. Autism Research 5: 385–397. PubMed ID: 23055267
  • Hodge JC, Mitchell E, Pillalamarri V, Toler TL, Bartel F, Kearney HM, Zou YS, Tan WH, Hanscom C, Kirmani S, Hanson RR, Skinner SA, et al. 2013. Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities. Molecular Psychiatry. PubMed ID: 23587880
  • Kleefstra, T. et al. (2012). "Disruption of an EHMT1-Associated Chromatin-Modification Moduce Causes Intellectual Disability." Am J Hu Genet 91(1):73-82. PubMed ID: 22726846
  • Laget S, Joulie M, Masson F Le, Sasai N, Christians E, Pradhan S, Roberts RJ, Defossez P-A. 2010. The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA. PLoS ONE 5: e11982. PubMed ID: 20700456
  • Mullegama SV, Rosenfeld JA, Orellana C, Bon BWM van, Halbach S, Repnikova EA, Brick L, Li C, Dupuis L, Rosello M, Aradhya S, Stavropoulos DJ, et al. 2013. Reciprocal deletion and duplication at 2q23.1 indicates a role for MBD5 in autism spectrum disorder. European Journal of Human Genetics. PubMed ID: 23632792
  • Talkowski ME, Mullegama SV, Rosenfeld JA, Bon BWM van, Shen Y, Repnikova EA, Gastier-Foster J, Thrush DL, Kathiresan S, Ruderfer DM, Chiang C, Hanscom C, et al. 2011. Assessment of 2q23.1 Microdeletion Syndrome Implicates MBD5 as a Single Causal Locus of Intellectual Disability, Epilepsy, and Autism Spectrum Disorder. The American Journal of Human Genetics 89: 551–563. PubMed ID: 21981781
  • Wagenstaller J, Spranger S, Lorenz-Depiereux B, Kazmierczak B, Nathrath M, Wahl D, Heye B, Gläser D, Liebscher V, Meitinger T, Strom TM. 2007. Copy-Number Variations Measured by Single-Nucleotide–Polymorphism Oligonucleotide Arrays in Patients with Mental Retardation. The American Journal of Human Genetics 81: 768–779. PubMed ID: 17847001
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TEST METHODS

Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution, and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as PCR, aCGH or MLPA before they are reported.
Analytical Validity

NextGen Sequencing

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect CNVs due to somatic mosaicism is limited.
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Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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