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Cornelia de Lange Syndrome Sequencing Panel

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1955 AFF4 81479 Add to Order
ANKRD11 81479
HDAC8 81479
KMT2A 81479
NIPBL 81479
RAD21 81479
SMC1A 81479
SMC3 81479
Full Panel Price* $640
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1955 Genes x (8) $640 81479(x8) Add to Order
Pricing Comments

We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

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

Over 70% of all CdLS patients harbor a pathogenic variant in NIPBL, SMC3, RAD21, SMC1A, or HDAC8 (Boyle et al. 2015). Only a few patients have been reported with pathogenic variants in AFF4, ANKRD11 and KMT2A.

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CNV via aCGH

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
600 ANKRD11$990 81479 Add to Order
NIPBL$990 81479
SMC1A$990 81479
SMC3$990 81479
Full Panel Price* $1190
Pricing Comments

# of Genes Ordered

Total Price

1

$990

2-5

$1190

6-10

$1290

11-100

$1490

Over 100

Call for quote
Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Intragenic NIPBL deletions and a duplication were identified in 13 (2.5%) out of 510 CdLS cases (12 deletions and 1 duplication) (Cheng et al. 2014). Large deletions and duplications account for 11% of reported SMC1A pathogenic variants (Gilissen et al. 2014; Baquero-Montoya et al. 2014). There are no reported large deletions or duplications in SMC3 (Human Gene Mutation Database). Deletion and duplication testing is currently unavailable for the HDAC8 and RAD21 genes. Large deletion/duplication accounts for ~30% of reported pathogenic variants in ANKRD11 (Human Gene Mutation Database).

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Clinical Features

Classic Cornelia de Lange syndrome (CdLS) is characterized by distinctive facial features, growth retardation, hirsutism, and upper limb reduction defects that range from subtle phalangeal abnormalities to oligodactyly. Craniofacial features include synophrys, arched eyebrows, long eyelashes, small upturned nose, small widely spaced teeth, and microcephaly. IQ ranges from below 30 to 102 (mean: 53). Many individuals demonstrate autistic and self-destructive tendencies. Frequent findings include cardiac septal defects, gastrointestinal dysfunction, hearing loss, myopia, and cryptorchidism or hypoplastic genitalia. Individuals with a milder phenotype have less severe growth, cognitive, and limb involvement, but often have facial features consistent with CdLS (Deardorff et al. 2012). CHOPS syndrome is characterized by cognitive impairment, coarse facies, heart defects, obesity, pulmonary involvement, short stature, and skeletal dysplasia (Izumi et al. 2015). CHOPS syndrome clinically overlaps with Cornelia de Lange syndrome. KBG syndrome is characterized by macrodontia of central upper incisors, mental retardation, dysmorphic facial features, short statue, skeletal anomalies and global developmental delay, seizures, and intellectual disability. KBG clinically overlaps with Cornelia de Lange syndrome (Ockeloen et al. 2015; Parenti et al. 2016).

Genetics

Cornelia de Lange syndrome is caused by pathogenic variants in 8 genes: NIPBL, SMC3, RAD21, SMC1A, HDAC8, KMT2A, AFF4, and ANKRD11. NIPBL, SMC3, RAD21, KMT2A, AFF4 and ANKRD11-related CdLS are all inherited in autosomal dominant manner, while SMC1A and HDAC8-related CdLS are inherited in an X-linked manner. Female carriers of SMC1A and HDAC8 pathogenic variants may show variable symptoms depending on random X-inactivation (Hoppman-Chaney et al. 2012; Mannini et al. 2013). The NIPBL, SMC3, RAD21, SMC1A, and HDAC8 genes encode regulatory or structural components of the cohesin complex, which play a key role involving sister chromatid cohesion, DNA repair, gene regulation, and genome stability (Barbero 2013). The KMT2A protein (Histone-lysine N-methyltransferase 2A) coded by exons 1 to 36 of the KMT2A gene on 11q23.3 is a member of H3K4-specific methyltransferases that plays an essential role in early development and hematopoiesis (Shen et al. 2014). The AFF4 protein coded by the AFF4 gene is a key component of the super elongation complex that regulates transcriptional processes during embryogenesis (Izumi et al. 2015). Approximately 70% of all CdLS patients have germline pathogenic variants in one of five genes: NIPBL (60%), SMC1A (5%), HDAC8 (5%), and less than 1% in SMC3 and RAD21 (Mannini et al. 2013; Kaiser et al. 2014, Boyle et al. 2015). One study reported that NIPBL somatic mosaicism was found in 23% of buccal swab samples of clinically diagnosed CdLS patients, who did not have a detectable pathogenic variant in the five known CdLS genes in their blood sample (Huisman et al. 2013). Approximately 360 unique NIPBL pathogenic variants have been reported. They are: missense (23%), nonsense (15%), splicing (17%), small del/ins (33%), gross del/ins (12%), and one complex arrangement. 99% of NIPBL pathogenic variants occur de novo. It has been suggested that milder forms of CdLS are more likely to be caused by missense mutation, and more severe forms by truncating mutations (Gillis et al. 2004; Boyle et al. 2015). Approximately 30 unique HDAC8 pathogenic variants have been reported. They are: missense (67%), large del/ins (23%), nonsense (10%), splicing (6%), one small deletion (Human Gene Mutation Database; Kaiser et al. 2014). Approximately 15 unique SMC3 pathogenic variants have been reported. They are: missense (60%), nonsense (1), in-frame small deletion (33%) and one nonsense. 9 out of those 15 pathogenic variants occur de novo, which explain 1%–2% of CdLS-like phenotypes (Gil-Rodríguez et al. 2015). Approximately 50 unique SMC1A pathogenic variants have been reported. They are: missense (58%), small deletion (14%), large del/ins, and complex rearrangement (10%) and one splicing (Musio et al. 2006; Baquero-Montoya et al. 2014; Ansari et al. 2014). Less than 10 unique RAD21 pathogenic variants have been reported. They are: missense (2), splicing (1), small deletion (1), and large deletions (3) (Deardorff et al. 2012; Minor et al. 2014; Ansari et al. 2014).

KMT2A-related CdLS is inherited in autosomal dominant manner. Pathogenic KMT2A variants mainly cause Wiedemann-Steiner syndrome, which is characterized by hypertrichosis cubiti, short status, intellectual disability, and dysmorphic facial and skeletal features (Jones et al. 2012). To date, approximately 15 unique pathogenic variants in KMT2A have been documented; most pathogenic variants were found in patients with Wiedemann-Steiner syndrome and only one de novo truncating pathogenic variant was reported in one patient with CdLS. The pathogenic variants include missense (4), nonsense (4), small del/ins (5), splicing (1) and large deletion (1). De novo pathogenic variants were found in five of the six Wiedemann-Steiner syndrome cases (Jones et al. 2012; Human Gene Mutation Database).

AFF4-related CHOPS syndrome is inherited in an autosomal dominant manner. Only 3 unique de novo missense AFF4 variants were reported in 3 unrelated probands affected with CHOPS syndrome by exome sequencing (Izumi et al. 2015).

ANKRD11-related CdLS and KBG syndrome are inherited in an autosomal dominant manner. Almost 50 pathogenic variants were reported, mainly found in patients with KBG syndrome. Most of the variants are truncating (nonsense and small deletion/insertion). Missense and splice variants are rare. Large deletion/duplications account for ~30% of reported ANKRD11 pathogenic variants (HGMD). Only 4 unique, pathogenic variants were reported in patients affected with CdLS, they all de novo, truncating variants (nonsense and small deletion/duplication) (Ansari et al. 2014; Parenti I. et al. 2016).

Testing Strategy

For this NextGen test, the full coding regions, plus ~10 bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed below. 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 any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic and variants of unknown significance calls are confirmed by Sanger sequencing.

Indications for Test

Candidates for this test are patients with clinical features consistent with Cornelia de Lange syndrome.

Genes

Official Gene Symbol OMIM ID
AFF4 604417
ANKRD11 611192
HDAC8 300269
KMT2A 159555
NIPBL 608667
RAD21 606462
SMC1A 300040
SMC3 606062
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
CHOPS Syndrome via AFF4 Gene Sequencing with CNV Detection
Cornelia de Lange Syndrome via ANKRD11 Gene Sequencing with CNV Detection
Cornelia de Lange Syndrome via SMC1A Gene Sequencing with CNV Detection
Skeletal Disorders and Joint Problems Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Ansari M. et al. 2014. Journal of Medical Genetics. 51: 659-68. PubMed ID: 25125236
  • Baquero-Montoya C. et al. 2014. Clinical Genetics. 85: 446-51. PubMed ID: 23683030
  • Barbero J.L. 2013. The Application of Clinical Genetics. 6: 15-23. PubMed ID: 23882154
  • Boyle M.I. et al. 2015. Clinical Genetics. 88: 1-12. PubMed ID: 25209348
  • Cheng Y.W. et al. 2014. Molecular Genetics & Genomic Medicine. 2: 115-23. PubMed ID: 24689074
  • Deardorff M.A. et al. 2012. American Journal of Human Genetics. 90: 1014-27. PubMed ID: 22633399
  • Gil-Rodríguez M.C. et al. 2015. Human Mutation. 36: 454-62. PubMed ID: 25655089
  • Gilissen C. et al. 2014. Nature. 511: 344-7. PubMed ID: 24896178
  • Gillis L.A. et al. 2004. American Journal of Human Genetics. 75: 610-23. PubMed ID: 15318302
  • Hoppman-Chaney N. et al. 2012. American Journal of Medical Genetics. Part A. 158A: 193-8. PubMed ID: 22106055
  • Huisman S.A. et al. 2013. Journal of Medical Genetics. 50: 339-44. PubMed ID: 23505322
  • Human Gene Mutation Database (Bio-base).
  • Izumi K. et al. 2015. Nature Genetics. 47: 338-44. PubMed ID: 25730767
  • Jones W.D. et al. 2012. American Journal of Human Genetics. 91: 358-64. PubMed ID: 22795537
  • Kaiser F.J. et al. 2014. Human Molecular Genetics. 23: 2888-900. PubMed ID: 24403048
  • Mannini L. et al. 2013. Human Mutation. 34: 1589-96. PubMed ID: 24038889
  • Minor A. et al. 2014. Gene. 537: 279-84. PubMed ID: 24378232
  • Musio A. et al. 2006. Nature Genetics. 38: 528-30. PubMed ID: 16604071
  • Ockeloen C.W. et al. 2015. European Journal of Human Genetics. 23: 1270. PubMed ID: 26269249
  • Parenti I. et al. 2016. Clinical Genetics. 89: 74-81. PubMed ID: 25652421
  • Shen E. et al. 2014. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 369: PubMed ID: 25135975
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

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 often 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.

Analytical Validity

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.   

Analytical Limitations

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/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

In the case of duplications, aCGH will not determine the chromosomal location of the duplicated DNA. Most duplications will be tandem, but in some cases the duplicated DNA will be inserted at a different locus. This method will also not determine the orientation of the duplicated segment (direct or inverted).

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay is dependent upon the quality of the input DNA. In particular, highly degraded DNA will yield poor results.

Order Kits

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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