Multiple Carboxylase Deficiency (Juvenile Onset) via the BTD Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
520 BTD$580.00 81404 Add to Order
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 18 days.

Clinical Sensitivity

Pomponio et al. (Pediat Res 42:840-848, 1997) identified mutations on each BTD allele in all 30 biotinidase-deficient probands analyzed. The p.Arg538Cys and p.Cys33PhefsStop36 mutations accounted for over 50% of the mutant alleles in this study.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 BTD$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Multiple carboxylase deficiency (MCD) is an inborn error of metabolism resulting from defective biotin metabolism.  Juvenile (also called late) onset MCD (OMIM #253260) results from profound or partial deficiency of biotinidase, whereas early onset MCD (OMIM #253270) is caused by holocarboxylase synthetase deficiency.  Both forms of MCD are responsive to biotin therapy.  Making a distinction between the two types is difficult and relies largely on age of onset rather than clinical features.  Clinical signs of juvenile onset MCD typically appear after 3 months of age while early onset MCD is typically evident earlier than 3 months of age (Wolf et al. Ann Neurol 18:614-617, 1985).  In vitro enzyme studies are capable of distinguishing between the two disorders.  The earliest presenting sign is usually seizures, but other early non-specific symptoms include hypotonia, respiratory symptoms, developmental delay, and ataxia.  Eczema, alopecia, dermatitis, and skin infections are also common findings, and cutaneous presentations in conjunction with neurological symptoms greatly limit the differential diagnosis.  Clinical variability is documented.  Untreated patients with partial biotinidase deficiency may experience fewer and milder symptoms than patients with complete deficiency (Suormala et al. J Inher Metab Dis 13:76-92, 1990).  Asymptomatic adults with profound biotinidase deficiency have also been reported (Wolf et al. Am J Med Genet 73:5-9, 1997).


BTD-related multiple carboxylase deficiency (MCD) is an autosomal recessive disorder.  Mutations in the BTD gene (OMIM #609019) are the genetic cause of juvenile or late onset MCD.  Over 100 BTD mutations have been reported in children affected with profound biotinidase deficiency.  Most pathogenic variants are missense mutations although nonsense, small insertions and deletions, and splice site mutations have also been found.  Three mutations (p.Cys33PhefsStop36, p.[Ala171Thr;Asp444His], and p.Gln456His) are commonly found in biotinidase-deficient newborns ascertained by newborn screening in the USA (Cowan et al. Genet Med 12:464-470, 2010).  The p.Cys33PhefsStop36 mutation is found commonly in symptomatic patients (Norrgard et al. Pediatr Res 46:20-27, 1999).  One other mutation (p.Arg538Cys) is common among symptomatic children, but not among newborns with a positive biotinidase screen (Pomponio et al. Hum Genet 99:506-512, 1997).  The p.Asp444His mutation reduces enzyme activity ~50% and results in a partial biotinidase deficiency when present in trans with a severe mutation.  The worldwide carrier frequency for BTD mutations is thought to be 1 in 120 (Wolf et al. J Inherit Metab Dis 14:923-927, 1991).

Testing Strategy

Biotinidase is encoded by exons 1-4 of the BTD gene on chr 3p25.  Testing is accomplished by amplifying the coding exons and ~10 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy sequencing methods and a capillary electrophoresis instrument.  We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Patients suspected of having multiple carboxylase deficiency based on biochemical testing and/or clinical features.


Official Gene Symbol OMIM ID
BTD 609019
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Multiple Carboxylase Deficiency, Juvenile Onset 253260


Genetic Counselors
  • Cowan, T. M., (2010). "Technical standards and guidelines for the diagnosis of biotinidase deficiency." Genet Med 12(7): 464-70. PubMed ID: 20539236
  • Norrgard, K. J., (1999). "Mutations causing profound biotinidase deficiency in children ascertained by newborn screening in the United States occur at different frequencies than in symptomatic children." Pediatr Res 46(1): 20-7. PubMed ID: 10400129
  • Pomponio, R. J., (1997). "Arg538 to Cys mutation in a CpG dinucleotide of the human biotinidase gene is the second most common cause of profound biotinidase deficiency in symptomatic children." Hum Genet 99(4): 506-12. PubMed ID: 9099842
  • Pomponio, R. J., (1997). "Mutations in the human biotinidase gene that cause profound biotinidase deficiency in symptomatic children: molecular, biochemical, and clinical analysis." Pediatr Res 42(6): 840-8. PubMed ID: 9396567
  • Suormala, T. M., (1990). "Comparison of patients with complete and partial biotinidase deficiency: biochemical studies." J Inherit Metab Dis 13(1): 76-92. PubMed ID: 2109151
  • Wolf, B. (1991). "Worldwide survey of neonatal screening for biotinidase deficiency." J Inherit Metab Dis 14(6): 923-7. PubMed ID: 1779651
  • Wolf, B., (1985). "Biotinidase deficiency: initial clinical features and rapid diagnosis." Ann Neurol 18(5): 614-7. PubMed ID: 4073853
  • Wolf, B., (1997). "Profound biotinidase deficiency in two asymptomatic adults." Am J Med Genet 73(1): 5-9. PubMed ID: 9375914
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking 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.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb 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 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

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 10 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.

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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