Indications for Testing
Testing for BTD is included in newborn screening panels throughout the U.S. and in a number of other countries. Newborns with abnormal screening results should undergo further testing of biotinidase enzyme activity to confirm or exclude a diagnosis of BTD. Testing is also warranted in parents of infants who have inconclusive newborn screening results, and in older individuals with symptoms that suggest BTD. (See Serum Biotinidase Testing below.)
DNA testing for BTD is appropriate to confirm profound or partial BTD or carrier status, and in relatives of affected individuals when familial variants are known. (See Molecular Testing below.)
Newborn screening for BTD involves the use of direct enzyme assays to assess dried blood spots for biotinidase activity. Traditionally, colorimetric assays have been used, but commercial kits using fluorescence are now available as well. Some U.S. states simply report positive or negative results, whereas others use an established cutoff value and report the specific levels of enzymatic activity detected. However, newborn screening cannot differentiate between partial and profound deficiency. Follow-up testing is necessary if screening results are abnormal.
Serum Biotinidase Testing
Serum or plasma testing is useful as a follow-up approach to measure biotinidase enzyme activity in newborns with abnormal screening results, and on occasion, in parents of newborns with uncertain screening results because BTD is unlikely if parental results are normal. Biotinidase testing is also appropriate in older patients who present with clinical symptoms suggestive of BTD.
The ideal strategy for serum testing is to measure enzyme activity in a control sample from an unrelated person and compare this activity with the patient’s enzyme assay results. For newborns, parental samples are recommended in addition to the control sample. Any specimens used for comparison should be collected at the same time as the patient’s sample and sent with the patient sample. This approach helps control for preanalytic variables, such as sample mishandling or sample compromise, which might lead to misdiagnosis.
Serum Test Results Interpretation
|Biotinidase Enzyme Activity
|<10% of mean normal activity
|10-30% of mean normal activity
|50% of mean normal activity
||Patient may be heterozygous for variants causative for profound BTD or homozygous for variant most commonly associated with partial BTD (p.Asp444His); consider molecular testing for variant identification
|Source: Strovel, American College of Medical Genetics and Genomics, 2017
It is important to note that metabolic tests, such as urine organic acid analysis using gas chromatography/mass spectrometry, may detect BTD-related biochemical features. However, this testing is not recommended for diagnosis of BTD because results are often normal in patients with BTD. Furthermore, abnormal metabolic test results, such as increased 3-hydroxyisovalerate levels, are nonspecific. Therefore, in patients with metabolic abnormalities that suggest possible BTD, serum biotinidase activity testing is recommended.
Genetic testing is helpful to confirm partial or profound BTD as well as carrier status, and has high clinical sensitivity when used in combination with biotinidase enzyme activity testing.
Given the lack of standardization among enzyme assays and reference ranges used by different laboratories, and because specific genetic variants are clearly associated with enzyme deficiency, some groups recommend that molecular testing be performed (after enzymatic testing) in all patients with suspected BTD. Molecular testing of the BTD gene is used to confirm newborn screening results in some states. Genetic testing can be especially helpful to distinguish between patients who have partial BTD, which is generally associated with the p.Asp444His variant, and patients who may be heterozygous carriers of variants associated with profound BTD.
Molecular testing for BTD involves targeted analysis or complete sequencing to detect BTD gene mutations. DNA from dried blood spots used for newborn screening can be analyzed to detect common variants. When a common variant panel fails to yield definitive results, complete gene sequencing or deletion/duplication testing should be considered.
The variant database hosted by ARUP Laboratories (https://arup.utah.edu/database/BTD/BTD_display.php ) is a helpful resource that includes information about more than 200 variants that affect biotinidase. Common variants associated with BTD in Caucasians include c. 98_104d7i3 (G98del7ins3), c. 1612C>T (p.R538C), c. 1368A>C (p.Q456H), and c. 511G>A (p.A171T:D444H).