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Amelogenesis Imperfecta with Gingival Hyperplasia Syndrome and Amelogenesis Imperfecta with Renal Syndrome via the FAM20A Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
6925 FAM20A$690.00 81479 Add to Order
Pricing Comment
For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

FAM20A mutations were identified in all 19 clinically diagnosed enamel renal syndrome cases from two studies (Wang et al, 2013; Jaureguiberry et al. 2013). Analytical sensitivity should be high, because almost all reported FAM20A mutations are point mutations or small deletions and duplications, which can be detected by sequencing. One large 79 base pair deletion has been reported (Jaureguiberry et al. 2013).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FAM20A$690.00 81479 Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

No gross deletions/duplications large enough to be detected by PreventionGenetics' aCGH have been reported in FAM20A (Human Gene Mutation Database).

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Clinical Features

Amelogenesis imperfecta (AI) is a heterogeneous condition of enamel defects affecting both primary and permanent dentitions. Affected teeth are usually small, discolored, pitted or grooved, and prone to rapid wear and breakage. Based on clinical and radiographic features of the enamel defects as well as on the mode of inheritance pattern, AI has been divided into 14 different subtypes, which can be grouped into four major forms: hypoplastic, hypomaturation, hypocalcified, and hypomaturation-hypoplastic with taurodontism (Witkop et al. 1988). Hypoplastic AI shows reduced enamel volume with pits or grooves, but enamel is usually hard and translucent. Hypomaturation and hypocalcified AI have hypomineralized enamel with nearly normal matrix volume. Hypocalcified AI may present soft enamel which can be easily scraped away by attrition. Hypomaturation enamel is hard, but brittle and prone to breaking off. Hypomaturation-hypoplastic with taurodontism shows reduced, hypomineralized enamel with pits; in addition, molars or other teeth may present enlarged pulp chambers (Witkop et al. 1988; Crawford et al. 2007). AI and AI-related syndrome are currently known to be caused by mutations in the following genes: AMELX (Aldred et al. 1992), DLX3 (Price et al. 1998), ENAM (Mardh et al. 2002), KLK4 (Hart et al. 2004), MMP20 (Kim et al. 2005), FAM83H (Lee et al. 2008; Kim et al. 2008), WDR72 (El-Sayed et al. 2009), FAM20A (O'Sullivan et al. 2011), C4orf26 (Parry et al. 2012), ROGDI (Schossig et al. 2012), SLC24A4 (Parry et al. 2013), ITGB6 (Poulter et al. 2013; Wang et al. 2013), LAMB3 (Kim et al. 2013), CNNM4 (Parry et al. 2009) and NHS (Burdon et al. 2003). Enamel defects can also occur as syndrome disorders. For example, Kohlschütter–Tönz syndrome features enamel defects, psychomotor delay or regression and seizures caused by ROGDI mutations (Tucci et al. 2013); Nance-Horan syndrome (NHS) is characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation caused by mutations in the NHS gene (Burdon et al. 2003); Jalili Syndrome features autosomal-Recessive Cone-Rod dystrophy and amelogenesis Imperfecta caused by mutations in the CNNM4 gene (Parry et al. 2009); and mutations in the FAM20A gene cause amelogenesis imperfecta and gingival hyperplasia syndrome as well as amelogenesis imperfecta and renal syndrome (O’Sullivan et al. 2011; Wang et al. 2013).

Genetics

Mutations in the FAM20A gene cause autosomal recessive amelogenesis imperfecta with gingival fibromatosis syndrome and amelogenesis imperfecta with renal syndrome (O’Sullivan et al. 2011; Wang et al. 2013). The FAM20A protein coded by the FAM20A gene belongs to a protein kinase family that plays crucial roles in regulation of biomineralization process. FAM20A is expressed in ameloblasts, odontoblasts, and the parathyroid gland and is suggested to have roles in modulating biomineralization during enamel and blood vessel formation (Vogel et al. 2012). To date, more than 20 unique pathogenic variants have been reported. They are: 6 nonsense, 2 missense, 6 splicing, 11 small deletion and insertions (O’Sullivan et al. 2011; Wang et al. 2013; Kantaputra et al. 2013). Only one gross deletion of 79 base pairs was reported in a patient affected with amelogenesis imperfect with renal syndrome (Jaureguiberry et al. 2013).

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. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed 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.

This test provides full coverage of all coding exons of the FAM20A gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Candidates for this test are patients with symptoms consistent with autosomal recessive hypoplastic amelogenesis imperfect, gingival fibromatosis, nephrocalcinosis and the family members of patients who have known FAM20A mutations.

Gene

Official Gene Symbol OMIM ID
FAM20A 611062
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Amelogenesis Imperfecta Sequencing Panel with CNV Detection
Amelogenesis Imperfecta via the DLX3 Gene
Amelogenesis Imperfecta via the FAM83H Gene
Amelogenesis Imperfecta via the KLK4 Gene
Amelogenesis Imperfecta via the MMP20 Gene
Amelogenesis Imperfecta via the WDR72 Gene
Epidermolysis Bullosa with Pyloric Atresia via the ITGA6 Gene
Junctional Epidermolysis Bullosa via the LAMB3 Gene
Kohlschutter-Tonz syndrome (KTS) via the ROGDI Gene
Nephrolithiasis and Nephrocalcinosis Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Aldred MJ, Crawford PJ, Roberts E, Thomas NS. 1992. Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1). Hum. Genet. 90: 413–416. PubMed ID: 1483698
  • Burdon KP, McKay JD, Sale MM, Russell-Eggitt IM, Mackey DA, Wirth MG, Elder JE, Nicoll A, Clarke MP, FitzGerald LM, Stankovich JM, Shaw MA, et al. 2003. Mutations in a Novel Gene, NHS, Cause the Pleiotropic Effects of Nance-Horan Syndrome, Including Severe Congenital Cataract, Dental Anomalies, and Mental Retardation. Am J Hum Genet 73: 1120–1130. PubMed ID: 14564667
  • Crawford PJ, Aldred M, Bloch-Zupan A. 2007. Amelogenesis imperfecta. Orphanet Journal of Rare Diseases 2: 17. PubMed ID: 17408482
  • El-Sayed W, Parry DA, Shore RC, Ahmed M, Jafri H, Rashid Y, Al-Bahlani S, Harasi S Al, Kirkham J, Inglehearn CF, Mighell AJ. 2009. Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta. The American Journal of Human Genetics 85: 699–705. PubMed ID: 19853237
  • Hart PS. 2004. Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. Journal of Medical Genetics 41: 545–549. PubMed ID: 15235027
  • Human Gene Mutation Database (Bio-base).
  • Jaureguiberry G, Dure-Molla M De la, Parry D, Quentric M, Himmerkus N, Koike T, Poulter J, Klootwijk E, Robinette SL, Howie AJ, Patel V, Figueres M-L, et al. 2012. Nephrocalcinosis (Enamel Renal Syndrome) Caused by Autosomal Recessive  FAM20A  Mutations. Nephron Physiology 122: 1–6. PubMed ID: 23434854
  • Kantaputra PN, Kaewgahya M, Khemaleelakul U, Dejkhamron P, Sutthimethakorn S, Thongboonkerd V, Iamaroon A. 2014. Enamel-renal-gingival syndrome and FAM20A mutations. American Journal of Medical Genetics Part A 164: 1–9. PubMed ID: 24259279
  • Kim J, Simmer J, Hart T, Hart P, Ramaswami M, Bartlett J, Hu J. 2005. MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet 42: 271–275. PubMed ID: 15744043
  • Kim J-W, Lee S-K, Lee ZH, Park J-C, Lee K-E, Lee M-H, Park J-T, Seo B-M, Hu JC-C, Simmer JP. 2008. FAM83H Mutations in Families with Autosomal-Dominant Hypocalcified Amelogenesis Imperfecta. The American Journal of Human Genetics 82: 489–494. PubMed ID: 18252228
  • Kim JW, Seymen F, Lee KE, Ko J, Yildirim M, Tuna EB, Gencay K, Shin TJ, Kyun HK, Simmer JP, Hu JC-C. 2013. LAMB3 mutations causing autosomal-dominant amelogenesis imperfecta. J. Dent. Res. 92: 899–904. PubMed ID: 23958762
  • Lee S-K, Hu JC-C, Bartlett JD, Lee K-E, Lin BP-J, Simmer JP, Kim J-W. 2008. Mutational Spectrum of FAM83H: The C-Terminal Portion is Required for Tooth Enamel Calcification. Hum Mutat 29: E95–E99. PubMed ID: 18484629
  • Mårdh CK, Bäckman B, Holmgren G, Hu JC-C, Simmer JP, Forsman-Semb K. 2002. A nonsense mutation in the enamelin gene causes local hypoplastic autosomal dominant amelogenesis imperfecta (AIH2). Hum. Mol. Genet. 11: 1069–1074. PubMed ID: 11978766
  • O’Sullivan J, Bitu CC, Daly SB, Urquhart JE, Barron MJ, Bhaskar SS, Martelli-Junior H, Santos Neto PE dos, Mansilla MA, Murray JC, Coletta RD, Black GCM, et al. 2011. Whole-Exome Sequencing Identifies FAM20A Mutations as a Cause of Amelogenesis Imperfecta and Gingival Hyperplasia Syndrome. Am J Hum Genet 88: 616–620. PubMed ID: 21549343
  • Parry DA, Brookes SJ, Logan CV, Poulter JA, El-Sayed W, Al-Bahlani S, Harasi S Al, Sayed J, Raïf EM, Shore RC, Dashash M, Barron M, et al. 2012. Mutations in C4orf26, Encoding a Peptide with In Vitro Hydroxyapatite Crystal Nucleation and Growth Activity, Cause Amelogenesis Imperfecta. The American Journal of Human Genetics 91: 565–571. PubMed ID: 22901946
  • Parry DA, Mighell AJ, El-Sayed W, Shore RC, Jalili IK, Dollfus H, Bloch-Zupan A, Carlos R, Carr IM, Downey LM, Blain KM, Mansfield DC, et al. 2009. Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta. Am J Hum Genet 84: 266–273. PubMed ID: 19200525
  • Parry DA, Poulter JA, Logan CV, Brookes SJ, Jafri H, Ferguson CH, Anwari BM, Rashid Y, Zhao H, Johnson CA, Inglehearn CF, Mighell AJ. 2013. Identification of Mutations in SLC24A4, Encoding a Potassium-Dependent Sodium/Calcium Exchanger, as a Cause of Amelogenesis Imperfecta. The American Journal of Human Genetics 92: 307–312. PubMed ID: 23375655
  • Poulter JA, Brookes SJ, Shore RC, Smith CEL, Farraj L Abi, Kirkham J, Inglehearn CF, Mighell AJ. 2013. A missense mutation in ITGB6 causes pitted hypomineralized amelogenesis imperfecta. Human Molecular Genetics. PubMed ID: 24319098
  • Price JA, Wright JT, Kula K, Bowden DW, Hart TC. 1998. A common DLX3 gene mutation is responsible for tricho-dento-osseous syndrome in Virginia and North Carolina families. J Med Genet 35: 825–828. PubMed ID: 9783705
  • Schossig A, Wolf NI, Fischer C, Fischer M, Stocker G, Pabinger S, Dander A, Steiner B, Tonz O, Kotzot D, Haberlandt E, Amberger A, et al. 2012. Mutations in ROGDI Cause Kohlschütter-Tönz Syndrome. Am J Hum Genet 90: 701–707. PubMed ID: 22424600
  • Tucci A, Kara E, Schossig A, Wolf NI, Plagnol V, Fawcett K, Paisán-Ruiz C, Moore M, Hernandez D, Musumeci S. 2013. Kohlschütter–Tönz Syndrome: Mutations in ROGDI and Evidence of Genetic Heterogeneity. Human mutation 34: 296–300. PubMed ID: 23086778
  • Vogel P, Hansen GM, Read RW, Vance RB, Thiel M, Liu J, Wronski TJ, Smith DD, Jeter-Jones S, Brommage R. 2012. Amelogenesis Imperfecta and Other Biomineralization Defects in Fam20a and Fam20c Null Mice. Veterinary Pathology 49: 998–1017. PubMed ID: 22732358
  • Wang S-K, Aref P, Hu Y, Milkovich RN, Simmer JP, El-Khateeb M, Daggag H, Baqain ZH, Hu JC-C. 2013. FAM20A Mutations Can Cause Enamel-Renal Syndrome (ERS). PLoS Genet 9: PubMed ID: 23468644
  • Wang S-K, Choi M, Richardson AS, Reid BM, Lin BP, Wang SJ, Kim J-W, Simmer JP, Hu JC-C. 2013. ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta. Hum. Mol. Genet. ddt611. PubMed ID: 24305999
  • Witkop CJ. 1988. Amelogenesis imperfecta, dentinogenesis imperfecta and dentin dysplasia revisited: problems in classification. Journal of Oral Pathology & Medicine 17: 547–553. PubMed ID: 3150442
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TEST METHODS

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 ~20 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 covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed 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, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  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 ~20 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.
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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