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TOWNES-BROCKS SYNDROME VIA THE SALL1 GENE

Clinical Features

Townes-Brocks syndrome (TBS, OMIM#107480) is a multiple malformation syndrome characterized by the triad of imperforate anus, dysplastic ears frequently associated with sensorineural and/or conductive hearing impairment, and thumb malformations (triphalangeal thumbs, preaxial polydactyly, hypoplastic thumbs) without shortening of the radius. Foot malformations (flat feet, overlapping toes) and genitourinary malformations are also common. Renal impairment including end-stage renal disease, congenital heart disease and mental retardation may occur (Kohlhase. GeneReviews. 2007).

Citations

Genetics

Townes-Brocks Syndrome (TBS) is inherited in an autosomal dominant manner. Penetrance seems complete, but expressivity is highly variable. SALL1 is the only gene known to be associated with TBS; about half of cases are caused by de novo mutations. SALL1 encodes sal-like protein 1, a C2H2 zinc finger transcription factor of the SAL type. Sal-like protein 1 appears to be an essential developmental regulator, which interacts with SALL4 and is regulated by TBX5 (Harvey&Logan Development 133:1165–1173, 2006). The majority of reported mutations in SALL1 are nonsense and frameshift mutations that positioned in exon 2 of the gene and resulted in premature stop codons. Both haploinsufficiency and dominant-negative effects of mutated SALL1 protein have been proposed for the pathogenesis of TBS (Kohlhase GeneReviews 2007; Maquat et al. Nat Rev Mol Cell Biol 5:89–99, 2004; Borozdin et al. Hum Mutat 27:211–212, 2006; Kiefer et al. Hum Mol Genet. 12:2221–2227, 2003).

Citations
  • Borozdin, W., et.al. (2006). "Detection of heterozygous SALL1 deletions by quantitative real time PCR proves the contribution of a SALL1 dosage effect in the pathogenesis of Townes-Brocks syndrome." Hum Mutat 27(2): 211-2. PubMed ID: 16429401
  • Harvey, S. A., Logan, M. P. (2006). "sall4 acts downstream of tbx5 and is required for pectoral fin outgrowth." Development 133(6): 1165-73. PubMed ID: 16501170
  • Kiefer, S. M., et.al. (2003). "Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects." Hum Mol Genet 12(17): 2221-7. PubMed ID: 12915476
  • Kohlhase (2007). "Townes-Brocks Syndrome." PubMed ID: 20301618
  • Maquat, L. E. (2004). "Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics." Nat Rev Mol Cell Biol 5(2): 89-99. PubMed ID: 15040442

Testing Strategy

This test involves bidirectional sequencing using genomic DNA of all coding exons of the SALL1 gene plus ~20 bp of flanking non-coding DNA on each side. We will also sequence and single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

Candidates for this test are patients with clinical features consistent with Townes-Brocks syndrome, and family members of patients who have known SALL1 mutations.

Gene(s)

Offical Gene Symbol OMIM Id
SALL1 602218

Disease(s)

Name OMIM Id
Townes-Brocks Syndrome 107480

Contacts

Genetic Counselors

Geneticist(s)

Test Methods

Bi-directional Sanger Sequencing

Test Number Test Price CPT Code
835 SALL1 Sanger Sequencing $890 81479
100 SALL1 Targeted Familial Mutations - Single Exon Sequencing $250 81479
200 SALL1 Targeted Familial Mutations - Double Exon Sequencing $370 81479
300 SALL1 Targeted Familial Mutations - Triple Exon Sequencing $440 81479

Test Procedure

As required, DNA is extracted from the patient specimen using a 5 Prime ArchivePure DNA Blood Kit. PCR is used to amplify the indicated exons plus additional flanking intronic or other non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. Sequencing is performed separately in both the forward and reverse directions.

Clinical Sensitivity

Sequencing of SALL1 is predicted to detect disease mutations in up to 70% of individuals with the classic triad of malformations (Kohlhase GeneReviews 2007; Kohlhase et al. Am J Hum Genet 64:435-445, 1999; Marlin et al. Hum Mutat 14:377-386, 1999). Large deletions involving single or multiple exons have been reported to account for less than 5% cases (Borozdin et al. Hum Mutat 27:211-212, 2006); such deletions are generally not detected by sequence analysis.

Citations
  • Borozdin, W., et.al. (2006). "Detection of heterozygous SALL1 deletions by quantitative real time PCR proves the contribution of a SALL1 dosage effect in the pathogenesis of Townes-Brocks syndrome." Hum Mutat 27(2): 211-2. PubMed ID: 16429401
  • Kohlhase (2007). "Townes-Brocks Syndrome." PubMed ID: 20301618
  • Kohlhase, J., et.al. (1999). "Molecular analysis of SALL1 mutations in Townes-Brocks syndrome." Am J Hum Genet 64(2): 435-45. PubMed ID: 9973281
  • Marlin, S., et.al. (1999). "Townes-Brocks syndrome: detection of a SALL1 mutation hot spot and evidence for a position effect in one patient." Hum Mutat 14(5): 377-86. PubMed ID: 10533063

Analytical Validity

As of July 2013, we compared 7.3 megabases of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 9.5 years of our lab operation we have Sanger sequenced roughly 2,000 PCR amplicons (~ 1 megabase). No errors have been identified.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Turnaround Time

Maximum of 40 days, although many tests are completed in 2-3 weeks.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Number Test Price CPT Code
600 SALL1 Deletion/Duplication Testing via aCGH $990 81479

*Prices:  Allele copy number analysis of one gene                                              $ 990
               Allele copy number analysis of two genes                                            $1190
               Allele copy number analysis of three or more genes                          $1290 + $100 for every additional gene
               Reanalysis of existing aCGH for additional genes (up to 5 genes)     $ 690

Test Procedure

As required, DNA is extracted from the patient specimen using a Gentra PUREGENE kit. 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 hours at 65ºC.  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.

PreventionGenetics’ high density gene-centric (HDGC) aCGH is designed to have comprehensive coverage for both coding and non-coding regions for each targeted gene with very high density probe coverage.  The average probe spacing within each exon is 47 bp or a minimum of three probes per exon covering all targeted exons and UTRs.  The average probe spacing is 289 bp covering all intronic, 2kb upstream and downstream regions of each targeted gene.  In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage.  Therefore, PreventionGenetics’ aCGH enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene.

Clinical Sensitivity


Analytical Validity

PreventionGenetics’ high density gene-centric custom designed aCGH enables the detection of relatively small deletion and amplification mutations (down to ~300 bp) within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

Any copy number changes smaller than 300bps (within the targeted region) may not be detected by our array.

This array may not detect deletion and amplification mutations 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 happened 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.

Turnaround Time

Maximum of 40 days, although many tests are completed in 3-4 weeks.

Requisition Form

  • The first three pages of the requisition form must accompany all specimens.
  • Billing information is on the third page.
  • Specimen and shipping instructions are listed on the fourth page.
  • All testing must be ordered by a qualified healthcare provider.

Download the Requisition Form (pdf)

Specimen Types

Whole Blood

(Delivery accepted Monday - Saturday)

  • Collect 2-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube).  For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
  • If refrigerated, blood specimen is good 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 a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml.  For tests involving the sequencing of  more than three genes, send an additional 5 µg DNA per gene.  DNA may be shipped at room temperature.
  • Label the tube with the DNA concentration as well as the patient name, date of birth, and/or ID number.
  • Specify the composition of the solute.
  • We only accept genomic DNA for testing.  We do not accept products of whole genome amplification reactions or other amplification reactions.

Cell Culture

(Delivery accepted Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Ship at least two T25 flasks of confluent cells.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We do not culture cells.

Version: 1.3
Last Updated 09/04/2012