Our laboratory first demonstrated that late-onset multiple carboxylase deficiency was due to a deficiency of biotinidase activity (5
), characterized the variability in the clinical expression of the biotinidase deficiency (19
), developed a method to screen newborns for biotinidase deficiency using blood-soaked filter paper spots (22
), piloted the first newborn screening program for the disorder (23
). We subsequently isolated and cloned the human biotinidase gene (24
) characterized the genomic organization of the gene (25
), and identified over one hundred different mutations that cause profound biotinidase deficiency (26
). In the past many investigators, including our laboratory, have attempted to infer information about biotinidase deficiency by studying rats made biotin-deficient by feeding them biotin-depleted diets. However, this approach is obviously flawed because these animals still have normal biotinidase activity and retain the ability to recycle biotin. Our biotinidase-deficient mice now eliminate this obstacle.
We observed the ratio of BTD+/+, BTD+/− and BTD−/− genotypes for newborn pups of about 1:2:1 in animals bred from pairs of heterozygous mice. This is the expected ratio for an autosomal recessive trait. The observed ratio and normal gestational development also indicates that biotinidase deficiency does not adversely affect fertilization, implantation or embryonic development.
Individuals heterozygous for one biotinidase-deficient mutation allele and one normal allele have half the serum biotinidase activity compared to that of the normal individuals. We confirmed that wildtype mice had about twice the serum biotinidase activity and CRM to antibody prepared against human biotinidase compared to heterozygous mice and that deficient mice have essentially undetectable biotinidase activity. The deficient mice have no detectable CRM present in serum consistent with what is observed for individuals with profound biotinidase deficiency due to a null mutation and individuals who are heterozygous for such a mutation.
Furthermore, biotin content and PCC activities in liver extracts of wildtype and heterozygous mice were similar, whereas both the hepatic biotin content and PCC activity of deficient mice were markedly reduced. These results support that only one functional copy of the BTD gene is sufficient to maintain biotin content and PCC activity and is consistent with what is observed in individuals who are heterozygous for profound biotinidase deficiency.
Interestingly, urinary excretion of biotin and biotinylated metabolites (BBM) was significantly greater in biotinidase-deficient mice compared to that of both the wildtype and heterozygous mice after 3–5 days of being fed the biotin-depleted diet. This is similar to what is observed in humans with biotinidase deficiency when withdrawn from biotin supplementation (27
). On the contrary, the mice with normal biotinidase activity when fed a biotin-depleted diet have decreased urinary biotin excretion (29
). The results indicate that the biotinidase, known to play a central role in intestinal absorption of biotin at the brush-border, may also have a role in renal reabsorption of the vitamin.
Neither wildtype nor heterozygous mice exhibited symptoms when placed on a biotin-deficient diet for the length of time necessary to elicit symptoms in deficient mice. This is consistent with what is observed in humans; heterozygous carrier individuals who have one defective and one functional copy of the BTD gene do not exhibit symptoms of the disorder.
Biotinidase-deficient mice exhibited growth delay and dramatic weight loss after being fed a biotin-deficient diet. They also exhibited hypotonia, deterioration of motor-neuron function, both hindlimb, forelimb and tail weakness, graying of fur with eventual loss of fur after about two weeks on the diet. All of the deficient mice became lethargic and began to limp with a hunched-up posture and stunted body frame, whereas heterozygous mice did not show any of these abnormalities and continued to grow normally.
In addition, bone volume of symptomatic deficient mice was decreased by 33 % compared to that of asymptomatic mice. Skeletal malformation and shortening of the long bones has been reported in fetuses of biotin-deficient dams (30
). We did not observe any bone malformations or defects nor limbs deformations in the heterozygous animal.
Biotin deficiency due to less than adequate dietary biotin in individuals with biotinidase deficiency usually leads to multiple carboxylase deficiency and subsequently to the accumulation of abnormal organic acids. In particular, decreased activity of 3MCG shunts the 3-methylcrotonyl-CoA to alternate metabolic pathways resulting in the accumulation of 3HIVA, 3MCG, and isovalerylglycine (IVG). In humans, urinary excretion of 3HIVA is considered the earliest, most sensitive indicator of biotin deficiency (31
), whereas 3-HPA and 2MCA are not (33
). Although it was experimentally challenging to obtain sufficient urine samples for analysis from symptomatic deficient mice, we found a 20-fold increase in the urinary excretion of 3HIVA in these mice compared to that of asymptomatic biotin-replete deficient mice, whereas the other organic acids were not elevated.
Many symptomatic children with biotinidase deficiency exhibit metabolic acidosis; however, this is not always present. In fact, we have found that symptomatic individuals often have elevated lactate and organic acid accumulation in the cerebrospinal fluid or by MRI spectroscopy, whereas there is no lactic acidemia/uria (34
). We did not find reduced serum bicarbonate or an increased anion gap in symptomatic deficient mice compared to deficient asymptomatic mice. We did observe mild hyperammonemia in symptomatic deficient mice, whereas the ammonia concentrations in asymptomatic deficient and normal mice were not elevated.
We attempted a pharmacological biotin rescue of symptomatic deficient mice by maintaining them on a biotin-deficient diet and supplementing them with pharmacological doses of biotin. The symptomatic deficient mice were treated with biotin injections, whereas symptomatic deficient mice and one heterozygous mouse received normal saline injections. Within several days of administering supplemental biotin, the symptomatic deficient mice showed marked improvement, whereas the symptomatic animals administered normal saline in place of biotin continued to deteriorate and lose weight. The biotin-treated deficient mice became more energetic and active and began to gain weight. In addition, these animals eventually grew back their body fur. This is similar to the reversibility of symptoms observed when symptomatic biotinidase-deficient children are diagnosed and treated with biotin.
We have clearly demonstrated that the transgenic, profoundly biotinidase-deficient mouse exhibits many of the clinical and biochemical features of untreated biotinidase deficiency in humans. In addition, biotin supplementation is able to reverse the clinical features of the disorder. Although many other enzyme-deficient mouse models have not met all the expectation for their usefulness in studying the pathophysiology or clinical features of the disorder seen in humans, our initial characterization of the biotinidase-deficient mouse is promising. Following further characterization, this animal model is likely to be useful in answering questions about biotinidase deficiency in a systematic and controlled environment. We will use this animal to study and compare the effect of biotin-restriction on the cutaneous, neurological (specifically auditory and ophthalmological) and immunological systems because these organ systems are affected in untreated children with biotinidase deficiency. It is important for us to determine if these mice develop sensorineural hearing loss and myelination defects, such as optic atrophy. We will also be able to study the potential toxicity of biocytin in the untreated and treated state.
In summary, the observations presented in this study indicate that our transgenic, knock-out mouse with biotinidase deficiency exposed to a biotin-deficient diet exhibits many of the clinical and biochemical characteristics of untreated children with biotinidase deficiency. Moreover, symptomatic, biotinidase-deficient mice markedly improve following treatment with pharmacological doses of biotin. This enzyme-deficient animal should provide important information about the disorder that cannot be obtained by studying individuals with the enzyme deficiency, especially now that fewer children with biotinidase deficiency will be available because of universal newborn screening.