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Prevention Genetics #861 ENG Sequencing
Messagehttp://preventiongenetics.com/clinical-dna-testing/requisition-forms/
Test Code
861
Preferred Specimen
Whole Blood 5 mL
Minimum Volume
2 mL
Other Acceptable Specimens
"ACD (yellow top tube). (DNA, Cell Culture) 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."
(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."
Instructions
For small babies, we require a minimum of 1 ml of blood
Transport Container
EDTA (purple top tube)
Transport Temperature
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 we
Specimen Stability
"At room temperature, blood specimen is good for up to 48 hours.
If refrigerated, blood specimen is good for up to one week
"
If refrigerated, blood specimen is good for up to one week
"
Reject Criteria (Eg, hemolysis? Lipemia? Thaw/Other?)
Frozen, insufficient quantity
Methodology
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
Setup Schedule
Delivery accepted Monday - Saturday
Report Available
Maximum of 40 days, although many tests are completed in 2-3 weeks
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."
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."
Clinical Significance
HHT is an autosomal dominant disorder caused by mutations in genes encoding proteins that modulate the normally inhibitory transforming growth factor (TGF)-ß signaling pathway during cell proliferation and differentiation. The incidence of HHT is ~ 1:5-8,000. HHT affects men, women and all ethnic groups (Govani and Shovlin Eur J Hum Genet 17:860-871, 2009). Mutations in the ENG gene (OMIM 131195) account for ~50-60% of HHT cases (HHT1; OMIM 187300). ENG encodes the endothelial cell surface co-receptor endoglin that binds (TGF)-ß and is essential for vascular integrity (Ríus et al. Blood 92:4677-4690,1998). HHT1 is associated with a high incidence of pulmonary and cerebral AVMs and a higher penetrance than HHT2 in which hepatic AVMs are more common (Letteboer et al. J Med Genet 43:371-377, 2006). HHT1 is thought to have a more severe phenotype than other forms of HHT. Causative mutations are found throughout the ENG gene and include primarily missense/nonsense mutations. Large whole or multi-exon deletions are also common (Prigoda et al. J Med Genet 43:722-728, 2006) as are splice site mutations and insertions. No predominant mutation has been identified.
