Craniosynostosis and Related Disorders Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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NextGen Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1957 FGFR1 81405 Add to Order
FGFR2 81479
FGFR3 81479
TCF12 81479
TWIST1 81404
Full Panel Price* $640.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1957 Genes x (5) $640.00 81404, 81405, 81479(x3) 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

~61% (111/182) of 182 Spanish craniosynostosis probands harbor a pathogenic variant in one of the FGFR2, FGFR3, TWIST1 and TCF12 genes. Pathogenic variants in FGFR2, FGFR3, TWIST and TCF12 account for 36%, 16%, 8% and 3% of pathogenic variants identified in this study, respectively (Paumard-Hernández et al. 2014). TCF12 explains 32% and 10% of patients affected with bilateral and unilateral Craniosynostosis, respectively (Sharma et al. 2013). One study reported that six unique FGFR1 pathogenic missense variants were found in seven unrelated patients affected with Hartsfield syndrome (Simonis et al. 2013). FGFR1 explains 5% of Pfeiffer syndrome type 1 cases (Robin et al. 2011).

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Del/Dup via aCGH

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
600 FGFR1$990.00 81479 Add to Order
FGFR2$990.00 81479
FGFR3$990.00 81479
Full Panel Price* $1190.00
Pricing Comments

# of Genes Ordered

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Over 100

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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Only five documented pathogenic variants in FGFR2 are large deletions/insertions (Human Gene Mutation Database; Bochukova et al. 2009). To date, no gross deletions or duplications have been reported in FGFR3 (Human Gene Mutation Database).

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Clinical Features

Craniosynostosis is a primary abnormality of premature fusion of the cranial sutures causing skull deformity, which can occur as non-syndromic or syndromic craniosynostosis with an approximate prevalence of 1 in 2,000 to 2,500 live births worldwide. Craniosynostosis related disorders include, but are not limited to the following disorders:

Achondroplasia is characterized by abnormal bone growth that results in short stature with disproportionately short arms and legs, a large head, and characteristic facial features with frontal bossing and mid-face retrusion (Pauli 2012).

Skeletal features of Hypochondroplasia are similar to achondroplasia, but are usually milder and show failure to grow as toddlers or school-age (Bober et al. 2013).

Thanatophoric dysplasia is a perinatal lethal short-limb dwarfism syndrome, which is divided into two subtypes: type I is characterized by micromelia with bowed femurs and usually without cloverleaf skull deformity; and type II is characterized by micromelia with straight femurs, and moderate-to-severe cloverleaf skull deformity (Karczeski and Cutting 2013). Other features of thanatophoric dysplasia are: short ribs, narrow thorax, macrocephaly, distinctive facial features, brachydactyly, hypotonia, and redundant skin folds along the limbs. Most affected infants die of respiratory insufficiency shortly after birth. Rare long-term survivors have been reported.

Crouzon syndrome is characterized by hypertelorism, exophthalmos and external strabismus, parrot-beaked nose, short upper lip, hypoplastic maxilla, and a relative mandibular prognathism (Vulliamy et al. 1966).

Apert syndrome is characterized by craniosynostosis, midface hypoplasia, and syndactyly of the hands and feet with a tendency to fusion of bony structures (Glaser et al. 2003).

CATSHL syndrome is characterized by camptodactyly, tall stature and hearing loss; some less common features include kyphoscoliosis, mental retardation, learning disabilities, and microcephaly (Toydemir et al. 2006).

Muenke syndrome is characterized by uni- or bicoronal synostosis, macrocephaly, midfacial hypoplasia, and developmental delay, other features include temporal bossing; widely spaced eyes, ptosis or proptosis (usually mild); midface retrusion; and highly arched palate or cleft lip and palate. Strabismus is common (Agochukwu et al. 2014). Trigonocephaly is a disorder caused by premature closure of the metopic sutures (Azimi et al. 2002).

Pfeiffer syndrome is characterized by coronal craniosynostosis, midface hypoplasia, and broad and medially deviated thumbs and great toes (Robin et al. 2011). Osteoglophonic dysplasia is characterized by rhizomelic dwarfism with depression of the nasal bridge, frontal bossing, and prognathism (Farrow 2006).

Jackson-Weiss syndrome is characterized by premature fusion of the cranial sutures and radiographic anomalies of the feet, and normal hands (Heike et al. 2001; Cohen et al. 2001).

Hartsfield syndrome is characterized by holoprosencephaly, ectrodactyly, with or without cleft/lip palate. Some patients may also present profound mental retardation and midline and limb defects (Vilain et al. 2009).

LADD syndrome (also called Levy-Hollister Syndrome) is the short name of Lacrimoauriculodentodigital syndrome, which is featured by abnormalities of the nasal lacrimal ducts, cup-shaped pinnas with mixed hearing deficit, small and peg-shaped lateral maxillary incisors and mild enamel dysplasia and fifth finger clinodactyly, duplication of the distal phalanx of the thumb, triphalangeal thumb, and syndactyly (Thompson et al. 1985).

Saethre-Chotzen syndrome is a craniosynostosis with low frontal hairline, facial asymmetry, brachydactyly, fifth finger clinodactyly, partial syndactyly, and vertebral column defects (Reardon and Winter 1994).

Bent bone dysplasia syndrome is characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones (Merrill et al. 2012).


Hartsfield syndrome, caused by pathogenic variants in FGFR1, is inherited in both an autosomal dominant and recessive manner (Simonis et al. 2013), while FGFR1-related Pfeiffer syndrome, Trigonocephaly, Osteoglophonic dysplasia, Jackson-Weiss syndrome and Kallmann syndrome are inherited in an autosomal dominant manner. FGFR1 protein encoded by FGFR1 is a growth factor receptor and a member of the FGFR family. Like all of the FGFRs, FGFR1 is a membrane-spanning tyrosine kinase receptor with an extracellular ligand-binding domain consisting of three immunoglobulin subdomains, a transmembrane domain, and a split intracellular tyrosine kinase domain (Green et al. 1996). To date, more than 150 unique causative variants have been reported in the FGFR1 gene. These variants are: missense (70%), nonsense (7%), splicing (7%), small insertion/deletions (13%) and only 4 gross deletions and genomic complex rearrangements (Human Gene Mutation Database). The majority (~90%) of reported pathogenic variants in the FGFR1 gene are found in patients affected with Kallmann or Kallmann-related disorders (Human Gene Mutation Database). Six of the missense variants were found in patients affected with Hartsfield syndrome. Only a few disease causing FGFR1 variants were reported in other FGFR1-related disorders, such as the c.755C>G (p.Pro252Arg) variant reported to cause Pfeiffer syndrome.

FGFR2-related disorders are inherited in an autosomal dominant manner. FGFR2 protein encoded by FGFR2 is a growth factor receptor, a member of the FGFR family. Like all of the FGFRs, FGFR2 is a membrane-spanning tyrosine kinase receptor with an extracellular ligand-binding domain consisting of three immunoglobulin subdomains, a transmembrane domain, and a split intracellular tyrosine kinase domain (Green et al. 1996). To date, more than 100 unique causative variants have been reported in the FGFR2 gene. These variants are: missense (66%), splicing (13%), small insertion/deletions (18%) and only 5 gross deletions and genomic complex rearrangements (Human Gene Mutation Database; Bochukova et al. 2009). Some genotype-phenotype correlations and recurrent pathogenic variants in FGFR2 have been documented, such as p.Ser252Trp and p.Pro253Arg which are responsible for 98% of Apert syndrome cases (Bochukova et al. 2009), while p.Cys342Tyr and p.Cys342Arg are often seen in Pfeiffer or Crouzon syndrome (Rutland et al. 1995; Mulvihill et al. 1995). For Crouzon syndrome or Pfeiffer syndrome, ~80% of FGFR2 pathogenic variants are located in exons 8 and 10, and ~10% of them are in exons 3, 5, 11, 14, 15, 16, and 17 (Robin et al. 2011). FGFR2 pathogenic  variants were found in 100% patients (227 patients) with Apert syndrome: 223/227 with point mutations and 4/227 with an Alu insertion or exon deletion (Bochukova et al. 2009).

All FGFR3-related disorders are inherited in an autosomal dominant manner, and the majority of cases result from de novo pathogenic variants caused by gain of functional variants. An exception is FGFR3-related CATSHL syndrome,which can be inherited either in an autosomal dominant manner or autosomal recessive manner through loss of function variants in the FGFR3 gene. The FGFR3 gene encodes fibroblast growth factor receptor-3, a member of the FGFR family. Like all of the FGFRs, FGFR3 is a membrane-spanning tyrosine kinase receptor with an extracellular ligand-binding domain consisting of three immunoglobulin subdomains, a transmembrane domain, and a split intracellular tyrosine kinase domain (Green et al. 1996). Some genotype-phenotype correlations have been well established. For example, most cases of achondroplasia are caused by one of two variants (c.1138G>A, p.Gly380Arg /c.1138G>C, p.Gly380Arg) in exon 10 (Shiang et al. 1994; Bellus et al. 1995; Deng et al. 1996). ~70% of Hypochondroplasia cases are caused by two variants (c.1620C>A, p.Asn540Lys and c.1620C>G, p.Asn540Lys) in exon 13 (Bellus et al. 1995; Prinos et al. 1995). Thanatophoric dysplasia type II is caused by the pathogenic variant c.1948A>G (p.Lys650Glu) in exon 15 (Bellus et al. 2000); and Muenke syndrome is caused by the c.749C>G, p.Pro250Arg variant in exon 7. Crouzon syndrome with acanthosis nigricans is caused by the pathogenic variant c.1172C>A, p. Ala391Glu in exon 10. In addition, two variants were found in two large families with CATSHL syndrome: heterozygous c.1862G>A, p.Arg621His in a family with autosomal dominant inheritance of CATSHL syndrome (Toydemir et al. 2006) and homozygous c.1637C>A, p.Thr546Lys in a family with autosomal recessive inheritance of CATSHL syndrome, respectively (Makrythanasis et al. 2014).

TCF12-related Craniosynostosis is inherited in an autosomal dominant manner. The TCF12 protein coded by the TCF12 gene is a member of the class A basic helix-loop-helix family, a partner of TWIST1 protein, which may be involved in the initiation of neuronal differentiation. Approximately 40 TCF12 pathogenic variants have been reported. They are: missense (7%), nonsense: (32%), splicing (17%), small deletion/insertions (41%), and one unbalanced translocation involving the TFC12 gene (Sharma et al. 2013; di Rocco et al. 2014; Le Tanno et al. 2014; Human Gene Mutation Database).

Saethre-Chotzen syndrome is inherited in an autosomal dominant manner. The TWIST1 protein coded by the TWIST1 gene is a transcription factor in the helix-loop-helix family which regulates embryonic development of many organs. Currently, ~170 unique TWIST1 pathogenic variants have been reported. They are: missense (34%), nonsense: (15%), small deletion/insertions (32%), large deletions (11%), and translocation/inversions (6%) (Johnson et al. 1998; Kress et al. 2006; Human Gene Mutation Database).

See individual gene test descriptions for additional information on molecular biology of gene products.

Testing Strategy

This Next Generation Sequencing (NGS) panel involves the simultaneous sequencing of the FGFR1, FGFR2, FGFR3, TWIST1 and TCF12 genes that have been implicated in Craniosynostosis and Related Disorders. For this NGS panel, each coding exon plus ~10bp of flanking non-coding DNA are simultaneously sequenced. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, 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, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Indications for Test

Candidates for this test are patients with clinical and radiologic features consistent Craniosynostosis and Related disorders. This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals who are suspected of any of these disorders, especially if clinical diagnosis is unclear, and individuals who have been found to be negative by mutation analysis for single gene tests are also candidates.


Official Gene Symbol OMIM ID
FGFR1 136350
FGFR2 176943
FGFR3 134934
TCF12 600480
TWIST1 601622
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

FGFR1-Related Disorders via the FGFR1 Gene
FGFR2-Related Disorders via the FGFR2 Gene
FGFR3-Related Disorders via the FGFR3 Gene
Achondroplasia via the FGFR3 Gene, Exon 10
Craniosynostosis via the TCF12 Gene
Hypochondroplasia via the FGFR3 Gene
Saethre-Chotzen Syndrome Via the TWIST1 Gene
Thanatophoric Dysplasia (TD) via the FGFR3 Gene


Genetic Counselors
  • Agochukwu NB, Doherty ES, Muenke M. 2014. Muenke Syndrome. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301588
  • Azimi C, Kennedy SJ, Chitayat D, Chakraborty P, Clarke JTR, Forrest C, Teebi AS. 2002. Clinical and genetic aspects of trigonocephaly: A study of 25 cases. American Journal of Medical Genetics Part A 117A: 127–135. PubMed ID: 12567409
  • Bellus GA, Hefferon TW, Ortiz de Luna RI, Hecht JT, Horton WA, Machado M, Kaitila I, McIntosh I, Francomano CA. 1995. Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am. J. Hum. Genet. 56: 368-373. PubMed ID: 7847369
  • Bellus GA, McIntosh I, Smith EA, Aylsworth AS, Kaitila I, Horton WA, Greenhaw GA, Hecht JT, Francomano CA. 1995. A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypochondroplasia. Nat. Genet. 10: 357–359. PubMed ID: 7670477
  • Bellus GA, Spector EB, Speiser PW, Weaver CA, Garber AT, Bryke CR, Israel J, Rosengren SS, Webster MK, Donoghue DJ, Francomano CA. 2000. Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype. Am. J. Hum. Genet. 67: 1411–1421. PubMed ID: 11055896
  • Bober MB, Bellus GA, Nikkel SM, Tiller GE. 2013. Hypochondroplasia. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301650
  • Bochukova EG, Roscioli T, Hedges DJ, Taylor IB, Johnson D, David DJ, Deininger PL, Wilkie AOM. 2009. Rare mutations of FGFR2 causing apert syndrome: identification of the first partial gene deletion, and an Alu element insertion from a new subfamily. Hum. Mutat. 30: 204–211. PubMed ID: 18726952
  • Cohen MM. 2001. Jackson-Weiss syndrome. American journal of medical genetics 100: 325–329. PubMed ID: 11343324
  • Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. 1996. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84: 911-921. PubMed ID: 8601314
  • di Rocco F, Baujat G, Arnaud E, Rénier D, Laplanche J-L, Daire VC, Collet C. 2014. Clinical spectrum and outcomes in families with coronal synostosis and TCF12 mutations. European Journal of Human Genetics 22: 1413–1416. PubMed ID: 24736737
  • Farrow EG, Davis SI, Mooney SD, Beighton P, Mascarenhas L, Gutierrez YR, Pitukcheewanont P, White KE. 2006. Extended mutational analyses of FGFR1 in osteoglophonic dysplasia. American Journal of Medical Genetics Part A 140A: 537–539. PubMed ID: 16470795
  • Glaser RL, Broman KW, Schulman RL, Eskenazi B, Wyrobek AJ, Jabs EW. 2003. The Paternal-Age Effect in Apert Syndrome Is Due, in Part, to the Increased Frequency of Mutations in Sperm. Am J Hum Genet 73: 939–947. PubMed ID: 12900791
  • Green PJ, Walsh FS, Doherty P. 1996. Promiscuity of fibroblast growth factor receptors. Bioessays 18: 639–646. PubMed ID: 8760337
  • Heike C, Seto M, Hing A, Palidin A, Hu FZ, Preston RA, Ehrlich GD, Cunningham M. 2001. Century of Jackson-Weiss syndrome: Further definition of clinical and radiographic findings in “lost” descendants of the original kindred. American journal of medical genetics 100: 315–324. PubMed ID: 11343323
  • Human Gene Mutation Database (Bio-base).
  • Johnson D, Horsley SW, Moloney DM, Oldridge M, Twigg SR, Walsh S, Barrow M, Njølstad PR, Kunz J, Ashworth GJ, Wall SA, Kearney L, Wilkie AO. 1998. A comprehensive screen for TWIST mutations in patients with craniosynostosis identifies a new microdeletion syndrome of chromosome band 7p21.1. Am J Hum Genet 63: 1282-1293. PubMed ID: 9792856
  • Karczeski B, Cutting GR. 2013. Thanatophoric Dysplasia. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301540
  • Kress W, Schropp C, Lieb G, Petersen B, Büsse-Ratzka M, Kunz J, Reinhart E, Schäfer W-D, Sold J, Hoppe F, Pahnke J, Trusen A, Sörensen N, Krauss J. 2006. Saethre-Chotzen syndrome caused by TWIST 1 gene mutations: functional differentiation from Muenke coronal synostosis syndrome. Eur. J. Hum. Genet. 14: 39-48. PubMed ID: 16251895
  • Le Tanno P, Poreau B, Devillard F, Vieville G, Amblard F, Jouk P-S, Satre V, Coutton C. 2014. Maternal complex chromosomal rearrangement leads to TCF12 microdeletion in a patient presenting with coronal craniosynostosis and intellectual disability. Am. J. Med. Genet. 164: 1530–1536. PubMed ID: 24648389
  • Makrythanasis P, Temtamy S, Aglan MS, Otaify GA, Hamamy H, Antonarakis SE. 2014. A Novel Homozygous Mutation in FGFR3 Causes Tall Stature, Severe Lateral Tibial Deviation, Scoliosis, Hearing Impairment, Camptodactyly, and Arachnodactyly. Human Mutation 35: 959-963. PubMed ID: 24864036
  • Merrill AE, Sarukhanov A, Krejci P, Idoni B, Camacho N, Estrada KD, Lyons KM, Deixler H, Robinson H, Chitayat D, Curry CJ, Lachman RS, Wilcox WR, Krakow D. 2012. Bent Bone Dysplasia-FGFR2 type, a Distinct Skeletal Disorder, Has Deficient Canonical FGF Signaling. Am J Hum Genet 90: 550–557. PubMed ID: 22387015
  • Mulvihill JJ. 1995. Craniofacial syndromes: no such thing as a single gene disease. Nat. Genet. 9: 101–103. PubMed ID: 7719329
  • Pauli RM. 2012. Achondroplasia. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301331
  • Paumard-Hernández B, Berges-Soria J, Barroso E, Rivera-Pedroza CI, Pérez-Carrizosa V, Benito-Sanz S, López-Messa E, Santos F, García-Recuero II, Romance A, Ballesta-Martínez JM, López-González V, Campos-Barros A, Cruz J5, Guillén-Navarro E, Sánchez Del Pozo J, Lapunzina P, García-Miñaur S, Heath KE. 2014. Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants. European Journal of Human Genetics. PubMed ID: 25271085
  • Prinos P, Costa T, Sommer A, Kilpatrick MW, Tsipouras P. 1995. A common FGFR3 gene mutation in hypochondroplasia. Human molecular genetics 4: 2097–2101. PubMed ID: 8589686
  • Reardon W, Winter RM. 1994. Saethre-Chotzen syndrome. J Med Genet 31: 393–396. PubMed ID: 8064818
  • Robin NH, Falk MJ, Haldeman-Englert CR. 2011. FGFR-Related Craniosynostosis Syndromes. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301628
  • Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Hayward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF. 1995. Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat. Genet. 9: 173–176. PubMed ID: 7719345
  • Sharma VP, Fenwick AL, Brockop MS, McGowan SJ, Goos JAC, Hoogeboom AJM, Brady AF, Jeelani NO, Lynch SA, Mulliken JB, Murray DJ, Phipps JM, Sweeney E, Tomkins SE, Wilson LC, Bennett S, Cornall RJ, Broxholme J, Kanapin A; 500 Whole-Genome Sequences (WGS500) Consortium, Johnson D, Wall SA, van der Spek PJ, Mathijssen IM, Maxson RE, Twigg SR, Wilkie AO. 2013. Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet 45: 304-307. PubMed ID: 23354436
  • Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M, Winokur ST, Wasmuth JJ. 1994. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78: 335–342. PubMed ID: 7913883
  • Simonis N, Migeotte I, Lambert N, Perazzolo C, Silva DC de, Dimitrov B, Heinrichs C, Janssens S, Kerr B, Mortier G, Vliet G Van, Lepage P, Casimir G, Abramowicz M, Smits G, Vilain C. 2013. FGFR1 mutations cause Hartsfield syndrome, the unique association of holoprosencephaly and ectrodactyly. Journal of Medical Genetics 50: 585–592. PubMed ID: 23812909
  • Thompson E, Pembrey M, Graham JM. 1985. Phenotypic variation in LADD syndrome. J. Med. Genet. 22: 382-385. PubMed ID: 4078868
  • Toydemir RM, Brassington AE, Bayrak-Toydemir P, Krakowiak PA, Jorde LB, Whitby FG, Longo N, Viskochil DH, Carey JC, Bamshad MJ. 2006. A novel mutation in FGFR3 causes camptodactyly, tall stature, and hearing loss (CATSHL) syndrome. Am. J. Hum. Genet. 79: 935-941. PubMed ID: 17033969
  • Vilain C, Mortier G, Vliet G Van, Dubourg C, Heinrichs C, Silva D de, Verloes A, Baumann C. 2009. Hartsfield holoprosencephaly-ectrodactyly syndrome in five male patients: Further delineation and review. American Journal of Medical Genetics Part A 149A: 1476–1481. PubMed ID: 19504604
  • Vulliamy DG, Normandale PA. 1966. Cranio-facial Dysostosis in a Dorset Family. Arch Dis Child 41: 375–382. PubMed ID: 21032436
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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 (  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 extracted and 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. PreventionGenetics has established and verified this test's accuracy and precision.

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.

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

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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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.
  • 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.


(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.


(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.


(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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