From January 2003 to September 2016, 6 patients were diagnosed with BTD in our hospital (Table
1). They were all born to Chinese parents except that the father of patient 5 is an Indian. Three patients were diagnosed because of clinical illness, and the other three were identified by newborn screening.
Table 1
Finding in patients with biotinidase deficiency
Diagnosed by | Clinical | Clinical | Clinical | Newborn screen | Newborn screen | Newborn screen |
Biotinidase activity (nmol/min/dL) (% of control mean) | 12.5 (7.8%) | 15.4 (9.8%) | NA | 36.5 (23%) | 36.3 (23%) | 32.5 (20%) |
C5OH at newborn screening (μM) | NA | 0.17 | NA | 0.14 | 0.10 | 0.12 |
C5OH at presentation (μM) | 3.5 | 3.37 | 0.362 | – | – | – |
BTD mutations | c.460-1G > A/c.1382 T > C (p.V461D) | c.460-1G > A/c.1382 T > C (p.V461D) | c.1384delA (p.R462Gfs) homozygous | c.1250_1251TC > AG (p.V417E)/c.1306G > A (p.E436K) | c.1361A > G (p.Y454C)/c.1306G > A (p.E436K) | c.1250_1251TC > AG (p.V417E)* |
Urine GCMS | Elevation of 3-hydroxyisovalerate, 3-methylcrotonylglycine, lactate, pyruvate | Elevation of 3-hydroxyisovaleric acid | Elevation of 3-hydroxyisovaleric acid | NA | NA | No specific finding |
Seizure onset age | 3 m | 3 m | 2 m | – | – | – |
Respiratory problems | Apnea requiring BiPAP at night | Laryngomalacia | – | – | – | – |
Hearing loss | + | – | + | – | – | – |
Optic atrophy | + | – | NA | – | – | – |
Eczema | + | + | NA | − | − | – |
Alopecia | – | – | – | – | – | – |
Candidiasis | – | Diaper rash | NA | – | – | – |
Current status | 10y, developmental delay | 3y, normal development | Expired at 2y, developmental delay | 5 m, asymptomatic | 1 m, asymptomatic | NA |
Case description
Patient 1 was noted to have seizures and an elevated plasma lactate level (4.95 mM, normal < 2.2 mM) when she was 3 months old. At the age of 6 years, she could only sit with support, babbled, and had hearing impairment, optic nerve atrophy, sleep apnea, proximal type renal tubular acidosis, and seizures that were controlled by a ketogenic diet. A brain magnetic resonance imaging (MRI) study revealed diffuse high intensity of white matter on T2-weighted images and a decreased N-acetylaspartate-to-choline (NAA/CHO) ratio and presence of a lactate signal on magnetic resonance spectrometry (MRS). A muscle biopsy revealed abnormal mitochondria reminiscent of mitochondrial disease, but sequencing of mitochondrial DNA revealed no pathogenic variants. She had recurrent eczema-like skin lesions. Her correct diagnosis was made after the diagnosis of her younger brother. Currently she still had apnea and needed a bilevel positive airway pressure (BiPAP) respirator at night time. She also had hearing loss.
Patient 2, the younger brother of patient 1, had laryngomalacia and seizures at the age of 3 months after an episode of respiratory tract infection. At that time, generalized skin rash, hypotonia, stridor, and septic shock were noted. A brain MRS study revealed the presence of a lactate peak. MCD was then suspected because his DBS C5OH-carnitine level was elevated (3.37 μM, normal < 0.36 μM). In addition, low biotinidase activity (15.4 nmol/min/dL) was noted. After the diagnosis of BTD, biotin 5 mg/day was prescribed. Stabilization of vital signs, increased muscle tone and activity, and resolution of skin lesions were observed in one week. He is now 3 years old and is seizure-free with normal development. His sister, at 8 years of age, started to learn to walk and had verbal interactions with family members after biotin treatment for approximately 1.5 years.
Patient 3 had seizures since 2 months of age. She could not sit or turn over and had poor responses to stimuli at the age of 2 years, when she died. Her DBS C5OH-carnitine level was mildly elevated (0.362 μM, normal < 0.182 μM), but urine organic acid analysis revealed an elevation of 3-OH-isovaleric acid levels. 3-Methylcrotonyl-CoA carboxylase deficiency was suspected, but her symptoms were not relieved after leucine restriction and carnitine supplementation. She died at 2 years of age. Final diagnosis was made after death.
Patients 4, 5 and 6 were found to have low DBS biotinidase by newborn screening after screening 46,958 newborns in an 11-month period in our screening center. Biotin 5 mg/day has been prescribed for patient 4. Patients 4, 5 and 6 are currently asymptomatic.
Biochemical profiles
General laboratory tests are not informative in BTD. Elevation of lactic acid may be a clue for metabolic diseases. This finding was described in patient 1, who had a plasma lactate level of 4.95 mM (normal < 2.2 mM) when she was 3 months of age, and in patient 2 because of a lactate peak identified by a brain MRS study. Elevation of C5OH-carnitine is not a reliable biomarker for biotinidase deficiency. C5OH-carnitine levels were significantly elevated in patient 1 (3.53 μM, normal < 0.74 μM) at 6 years of age and in patient 2 (3.37 μM) at the age of 3 months. However, the C5OH-carnitine level was normal in patient 2 at birth by newborn screening and was only mildly elevated in patient 3 (0.362 μM) at the age of 2 years. Abnormal results of urine organic acid analysis were reported for patients 1–3 as an elevation of 3-OH-isovaleric acid levels with or without other characteristic abnormal organic acids in urine, including 3-methylcrotonylglycine, lactate and pyruvate, suggesting MCD.
Biotinidase activity
Biotinidase activities in the first two patients were very low: 15.4 nmol/min/dL (9.8% of control mean) and 12.5 nmol/min/dL (7.8% of control mean). However, the activities in the three patients detected by newborn screening were higher: 36.5 nmol/min/dL (23% of control mean), 36.3 nmol/min/dL (23% of control mean), 32.5 nmol/min/dL (20% of control mean), respectively.
Molecular analysis
Patients 1 and 2 had compound heterozygous mutations c.460-1G > T and c.1382 T > A (p.V461D) on the BTD gene, which were inherited from both parents. Mutation analysis after the death of patient 3 revealed a homozygous c.1384delA variant of the BTD gene. Patient 4 had compound heterozygous c.1250_1251TC > AG (p.V417E) and c.1306G > A (p.E436K) mutations. Patient 5 had c.1361A > G (p.Y454C)/ c.1306G > A (p.E436K) mutations. Patient 6 had c.1250_1251TC > AG (p.V417E) homozygous mutation or deletion. Two of the variants, c.1384delA and c.1361A > G, had been reported: the patient with homozygous c.1384delA had 13% biotinidase activity but suffered from apnea, unconsciousness, convulsions, hearing impairment, and mental retardation [
20]; the patient with compound heterozygous c.1361A > G mutation had profound biotinidase deficiency [
21]. The other four, c.460-1G > T, c.1382 T > A, c.1250_1251TC > AG, and c.1306G > A, are novel variants but are predicted as pathogenic or likely pathogenic (Table
2).
Table 2
List of mutations identified in the current study
c.460-1G > A | chr3:15685822 | NA | NA | NA | NA | NA | NA | NA | NA | NA | Pathogenic (PVS1 PM2 PM3) |
c.1250_1251TC > AG (p.V417E) | chr3:15686613-chr3:15686614 | rs750006399 / rs764811197 | NA | NA | NA | 0.0002718 (all East Asian) | 0.00001989 | 0.000989 | D | PD | Likely pathogenic (PM2 PM3 PP3 PP4) |
c.1306G > A (p.E436K) | chr3:15686669 | rs749460715 | NA | Likely pathogenic / VUS | NA | 0.0004510 (all East Asian) | 0.00003182 | 0.000989 | D | PD | Likely pathogenic (PM2 PM3 PP3 PP5) |
c.1361A > G (p.Y454C) | chr3:15686724 | rs397514345 | Pathogenic | Likely pathogenic / Pathogenic / VUS | DM | 0.002058 (all South Asian) | 0.0002625 | NA | D | PD | Likely pathogenic (PM2 PM3 PM5 PP3 PP5) |
c.1382 T > C (p.V461D) | chr3:15686745 | NA | NA | NA | NA | NA | NA | NA | T | B | Likely Pathogenic (PM2 PM3 PP3 PP4) |
c.1384delA (p.R462Gfs) | chr3:15686747–15,686,747 | rs397514420 | Pathogenic | Pathogenic | DM | 0.0001087 (all East Asian) | 0.000007955 | NA | NA | NA | Likely Pathogenic (PVS1 PM2 PM3 PP4 PP5) |