Background:
Craniosynostosis is the premature fusion (closure) of one or more sutures of the skull. It is a developmental anomaly with age of onset in utero and infancy. The early sutural closure often leads to abnormal head shape and facial dysmorphia. Craniosynostosis can occur in isolation (nonsyndromic) or as part of a larger syndrome with additional facial, skeletal, and organ anomalies (syndromic). There are approximately 200 syndromes associated with craniosynostosis. Clinical phenotype indicating a syndromic cause of craniosynostosis include limb defects, ear and eye abnormalities, maxillary and mandibular hypoplasia, and or cardiovascular malformations. It is a genetically heterogeneous disorder with mutations identified in multiple genes. Inheritance is autosomal dominant and recessive depending on syndrome. Chromosomal alterations are a causative mechanism of the syndromic forms of craniosynostosis in more than 10% of the cases.
Reasons for Referral:
- Patient displays craniofacial phenotype consistent with craniosynostosis
- Radiography and CT reveal premature fusion of one or more cranial sutures. Patient may one or more of the following: Chiari malformations, facial dysmorphia and or asymmetry, ptosis, blepharophimosis, hypertelorism, proptosis, down slanting palpebral fissures, choanal atresia or stenosis, flat nasal bridge, midfacial hypoplasia, maxillary hypoplasia, micrognathia, mandibular prognathism, oral abnormalities, radiohumeral synostosis, radioulnar synostosis, syndactyly of the fingers and toes, brachydactyly, broad halluces with medial deviation
- Carrier testing
- Positive family history
Methodology:
Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs 87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.
Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmental duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.
This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.
The 35 Craniosynostosis-associated genes are listed below:
ALPL, ALX4, ASXL1, CDC45, CYP26B1, EFNB1, ERF, FBN1, FGFR1, FGFR2, FGFR3, FREM1, GLI3, IFT122, IFT43, IL11RA, MASP1, MEGF8, MSX2, P4HB,PHEX, POR, RAB23, RECQL4, SEC24D, SKI, SLC25A24, TCF12, TGFBR1, TGFBR2, TMCO1, TWIST1, WDR19, WDR35, ZIC1
Specimen Requirements:
Blood: EDTA or ACD (Solution A or B):
- Adult: 5 mL
- Child: 5 mL
- Infant: 2-3 mL
Saliva: 2 ORAgene™ Saliva Collection Kit(s) (OGR-500). Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Assisted Saliva: 4 ORAgene™ Saliva Collection Kit(s) (OGR-575) used per manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Buccal Cells: 4 CytoSoft™ Cytology Brush (Medical Packaging CYB-1) used according to manufacturer instructions. Please contact KDL Client Services for a Buccal Collection Kit for patients that cannot provide a blood sample.
Skin Fibroblast: Punch Biopsy (cell cultures will be prepared at KDL and used for testing), or 2 T-25 confluent flasks.
Prenatal:
- Direct Amniotic Fluid (10-20mL)
- Direct CVS
- Direct POC
- Cultured Amniocytes (2 T-25 flasks)
- Cultured CVS (2 T-25 flasks)
- Cultured Fetal Tissue: Product of Conception (2 T-25 flasks)
- Cord Blood (1-2mL)
DNA: 10 µg at a minimum of 60-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).
Notice Regarding Molecular Genetic Testing on Prenatal Specimens:
Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood (or saliva) in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.
For routine testing of blood and saliva (or DNA extracted from them), KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed within the above windows, please contact our lab.
Test Performed (Days):
Weekly
Turn Around Time:
8 weeks
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold, but not frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
References:
- Wilke AO, Bochukova EG, Hansen RM, et al. Clinical dividends from the molecular genetic diagnosis of craniosynostosis. Am J Med Genet A. 2007; 143A(16):1941-98
- Panigrahi I. Craniosynostosis genetics: The mystery unfolds. Indian J Hum Genet. 2011; 17(2):48-53
- Agochukwu NB, Solomon BD, Muenke M. Impact of genetics on the diagnosis and clinical management of syndromic craniosynostosis. Childs Nerv Syst. 2012;28(9):1447-63
Additional Info: