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OFD1-Related Disorders via OFD1 Gene Sequencing with CNV Detection

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

Sequencing with CNV

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
11541 OFD1$890 81479,81479 Add to Order

New York State Approved Test

Pricing Comments

Our favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, if a lower cost sequencing option without CNV detection is desired, please see this link for Test Code, pricing, and turnaround time information. If the alternative option is selected, CNV detection may be ordered through Test #600.

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 26 days.

Clinical Sensitivity

Roughly 80% of females with OFD1 syndrome are found to have OFD1 pathogenic variant detected by sequencing (Prattichizzo et al. 2008). Approximately 5% of females with OFD1 syndrome will have a gross deletion (Thauvin-Robinet et al 2009). The clinical sensitivity for Joubert syndrome, Simpson-Golabi-Behmel syndrome, and retinitis pigmentosa is unknown due to the paucity of documented cases. Analytical sensitivity should be high for Joubert syndrome, Simpson-Golabi-Behmel syndrome, and retinitis pigmentosa since all OFD1 pathogenic variants reported to date are expected to be detected by sequencing.

Roughly 20% of individuals with OFD1 syndrome who have a negative finding by sequencing have been found to have a gross deletion in OFD1 (Thauvin-Robinet et al. 2009).

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Clinical Features

Oral-Facial-Digital syndrome type 1 (OFD1) is a developmental disorder with variable expressivity characterized by oral anomalies (lobed tongue, cleft palate, gingival frenulae, dental abnormalities); facial anomalies (ocular hypertelorism, hypoplasia of the alae nasi, upper lip median cleft, micrognathia); digital anomalies (brachydactyly, syndactyly, clinodactyly; preaxial or postaxial polydactyly, duplicated hallux); brain anomalies (intracerebral cysts, corpus callosum agenesis, cerebellar agenesis with or without Dandy-Walker malformation) and polycystic kidneys. In addition, about 50% of patients diagnosed with OFD1 have learning disabilities (Gorlin and Psaume 1962; Feather et al. 1997; Ferrante et al. 2001).

Joubert Syndrome (JBTS) is marked by mid-hindbrain malformation, retinal dystrophy, cystic renal disease, hepatic fibrosis and polydactyly (Doherty 2009). Mid-hindbrain malformation, which can be readily identified as a “Molar Tooth Sign” (MTS) using Magnetic Resonance Imaging (MRI), typically leads to hypotonia, ataxia, abnormal eye movements and intellectual disability. MTS is pathognomonic for JBTS.

Retinitis Pigmentosa is an inherited degenerative disease of the retina characterized by abnormalities of the photoreceptors or the retinal pigment epithelium. It is a progressive disease. Symptoms usually begin with night blindness, progressing to constriction of the peripheral visual field and, eventually, to loss of central vision. The age of onset varies from childhood to middle age (Gu et al. 1999).

Genetics

OFD1 is embryonic male-lethal X-linked dominant condition caused by pathogenic variants in the OFD1 gene (Feather et al. 1997; Ferrante et al. 2001). OFD1 encodes oral-facial-digital syndrome 1 protein (OFD1), which is localized to centrosomes and basal bodies of ciliated cells suggesting a role in cilia motility and function (Ferrante et al. 2009; Coene et al. 2009). A mix of missense, nonsense, frameshift and splicing variants as well as gross deletions have been reported in OFD1 (Ferrante et al. 2001; Rakkolainen et al. 2002; Morisawa et al. 2004; Budny et al. 2006; Coene et al. 2009). Roughly 75% of OFD1 cases are simplex (Toriello and Franco 2013). Severity of the phenotype correlates with the reduction in protein length (Thauvin-Robinet et al. 2006). Coene et al. predicted that all pathogenic variants before residue 631 are lethal for males and cause OFD I syndrome in females, while males with JBS10 who may live beyond the age of 30 years have pathogenic variants located in the coiled-coil domain nearest to the C terminus (Coene et al. 2009).

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 OFD1 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

We also sequence the intronic c.935+706A>G variant (Webb et al. 2012).

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Candidates for this test are patients with symptoms consistent with X-linked dominant OFD1, and X-linked recessive Simpson-Golabi-Behmel syndrome type 2, Joubert syndrome 10, and retinitis pigmentosa, and family members of patients who have known OFD1 variants.

Gene

Official Gene Symbol OMIM ID
OFD1 300170
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Budny B. et al. 2006. Human Genetics. 120: 171-8. PubMed ID: 16783569
  • Coene K.L. et al. 2009. American Journal of Human Genetics. 85: 465-81. PubMed ID: 19800048
  • Doherty D. 2009. Seminars in Pediatric Neurology. 16: 143-54. PubMed ID: 19778711
  • Feather S.A. et al. 1997. Human Molecular Genetics. 6: 1163-7. PubMed ID: 9215688
  • Ferrante M.I. et al. 2001. American Journal of Human Genetics. 68: 569-76. PubMed ID: 11179005
  • Ferrante M.I. et al. 2009. Human Molecular Genetics. 18: 289-303. PubMed ID: 18971206
  • Gorlin R.J., Psaume, J. 1962. The Journal of Pediatrics. 61: 520-30. PubMed ID: 13900550
  • Gu S. et al. 1999. Journal of Medical Genetics. 36: 705-7. PubMed ID: 10507729
  • Morisawa T. et al. 2004. Human Genetics. 115: 97-103. PubMed ID: 15221448
  • Prattichizzo C. et al. 2008. Human Mutation. 29: 1237-46. PubMed ID: 18546297
  • Rakkolainen A. et al. 2002. Journal of Medical Genetics. 39: 292-6. PubMed ID: 11950863
  • Thauvin-Robinet C. et al. 2006. Journal of Medical Genetics. 43: 54-61. PubMed ID: 16397067
  • Thauvin-Robinet C. et al. 2009. Human Mutation. 30: E320-9. PubMed ID: 19023858
  • Toriello H.V, Franco B. 2013. Oral-Facial-Digital Syndrome Type I. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301367
  • Webb T. R. et al. 2012. Human Molecular Genetics. 21: 3647-54. PubMed ID: 22619378
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

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.

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.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; 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., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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 amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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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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