Biotinidase deficiency (BTD) is an inherited disorder caused by biallelic pathogenic variants in the BTD gene and affects approximately one in 60,000 people. The deficiency in biotinidase enzymatic activity interferes with the body’s ability to recycle and reuse the vitamin biotin, resulting primarily in neurologic and dermatologic manifestations. The disorder usually causes no symptoms in the first weeks or months of life, and early signs and symptoms are frequently nonspecific. Symptoms can develop once the biotin transferred from the mother to the child has been depleted. Hearing impairment, vision problems, and developmental delay are generally irreversible once they have occurred, even with biotin therapy; therefore, timely diagnosis is very important. Early treatment can prevent all symptoms. Newborn screening for BTD is currently performed across the United States and in more than 30 other countries. Confirmatory testing following an abnormal newborn screen for BTD involves serum testing to evaluate biotinidase activity and may include molecular testing of the BTD gene. Laboratory testing may also be indicated in older patients who manifest symptoms that suggest the disease, and in cases of uncertain newborn screening results.
Quick Answers for Clinicians
Clinical presentation can vary significantly in patients with biotinidase deficiency (BTD), even among related individuals. BTD is classified as either profound or partial, depending on the degree of enzyme activity detected. Patients with profound untreated BTD may suffer from seizures, hypotonia, developmental delay, hearing and vision impairment, alopecia, and ataxia, among other conditions. Developmental delay and vision or hearing impairment may be permanent if they occur before treatment begins. Coma and death can also result from untreated profound BTD. Rarely, patients have profound deficiency without clinical manifestations. Patients with partial BTD who do not undergo treatment can be asymptomatic or may suffer from mild forms of the conditions associated with profound deficiency, but typically only in the context of a stressor such as surgery or infection.
Biotinidase testing is typically performed following an abnormal newborn screening result, but may be part of a broader workup in children with clinical characteristics of biotinidase deficiency (BTD). Testing should also be considered in individuals who manifest clinical symptoms of BTD following a stressor such as illness or surgery, and in those with symptoms of the disorder who may not have been screened at birth. On occasion, late-onset BTD in adolescents and adults has been mistaken for multiple sclerosis (MS); enzyme testing to rule out BTD should be considered in patients with clinical suspicion of MS.
When newborn screening test results for BTD are inconclusive or uncertain, parental testing may be helpful. For instance, screening results may be affected by blood transfusions in the neonate, and normal parental results can be used to help rule out BTD in such cases.
The level of enzyme activity detected in serum or plasma distinguishes partial from profound biotinidase deficiency (BTD). Patients with enzyme activity levels 10-30% of normal are considered to have partial deficiency, whereas those with levels <10% of normal are considered to have profound deficiency. In addition, specific mutations are causative for either partial or profound BTD. For example, four common pathogenic variants are associated with profound BTD, whereas the common c. 1330G>C (p.D444H) pathogenic variant is associated with partial deficiency. Therefore, molecular testing can be helpful to characterize whether the deficiency is profound or partial. See Genetic Testing for additional information.
Although specific levels of detected enzymatic activity are included in newborn screening test results in some U.S. states, newborn screening should not be used to differentiate between partial and profound biotinidase deficiency (BTD). False-positive BTD screening results (low levels of enzymatic activity) can occur in premature infants and as a result of specimen mishandling (eg, humidity reduces enzyme activity in samples). Therefore, follow-up testing is necessary in the event of abnormal screening results.
ARUP Laboratories hosts a publicly accessible database with additional information about more than 200 variants that affect biotinidase enzyme activity: https://arup.utah.edu/database/BTD/BTD_display.php. More than 190 of these variants are considered pathogenic.
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 Genetic 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.
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).
ARUP Lab Tests
Initial biotinidase enzyme test to diagnose or rule out BTD
Molecular DNA test to confirm a diagnosis of BTD when biotinidase enzymatic activity is low
Recommended test for a known familial sequence variant previously identified in a family member
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