Simple 3-methylcrotonyl coenzyme A carboxylase deficiency is unresponsive to biotin therapy and must be differentiated from biotin-responsive multiple carboxylase deficiency. The latter is due to impaired biotin metabolism, i.e., biotinase and total carboxylase synthase deficiency, involving all four biotin-dependent carboxylases. All diseases in this category have 3-methylcrotonyl coenzyme A carboxylase deficiency, and the major urinary metabolites are 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. In cases of multiple carboxylase deficiency there is also a small increase in methylcitrate and 3-hydroxypropionic acid, characteristic of crotonyl coenzyme A carboxylase deficiency. Therefore, the diagnosis of simple 3-methylcrotonyl coenzyme A carboxylase deficiency depends on quantitative analysis of urinary organic acid profile, or enzymatic examination of fibroblasts and leukocytes, as well as plasma biotinase assay, to determine whether this enzyme deficiency alone or other carboxylase deficiency is present. The characteristic abnormal metabolites of 3-methylcrotonyl coenzyme A carboxylase deficiency are 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, with extremely high urinary excretion of 460-5900 mmol/mole creatinine and 70-3700 mmol/mole creatinine, respectively. Other abnormal products may be 2-oxoglutarate and 3-methylcrotonyl glutamate. Patients have severe secondary plasma free carnitine deficiency and a significantly higher carnitine ester to free carnitine ratio, suggesting abnormal carnitine ester excretion, mainly 3-hydroxyisovaleryl carnitine. The activity of 3-methylcrotonyl coenzyme A carboxylase in cultured fibroblasts of the patient was significantly reduced to 0%-2% of normal, independent of biotin treatment, and the activity of other carboxylases was normal. This disorder is autosomal recessive. Clinical manifestations] Children with simple 3-methylcrotonyl coenzyme A carboxylase deficiency have normal growth and development before the first acute attack. The first attack is usually in the 14th to 33rd month of life, but can occur as early as ll weeks or as late as 5 years of age. The clinical presentation is similar to Reye’s syndrome or 3-hydroxy-3-methylglutaryl coenzyme A cleavage enzyme deficiency. It usually occurs after a mild infection with feeding difficulties, vomiting, lethargy, apnea, hypotonia or hyperreflexia, and may have myoclonus or convulsions. Some cases have neutrophilia, which may be associated with infection or nonspecific stress adrenaline release. Individual cases have alopecia, cardiac arrest, cerebral edema, or Reye’s syndrome-like fatty liver. Typical laboratory findings are severe hypoglycemia (blood glucose below lmM), hyperammonemia, increased hepatic transaminase activity, mild metabolic acidosis, and moderate ketonuria. The plasma free carnitine concentration is extremely low, 0.7-5 μM (normal >20 μM), and the carnitine ester ratio is increased. A few cases may have no clinical symptoms. 【Diagnosis】The disease should be considered if there are typical symptoms of organic aciduria, especially those showing significant hypoglycemia or Reye’s syndrome. Urinary excretion of large amounts of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine is diagnostic. An increase in these organic acids should not be accompanied by isovalerylglycine, 3-methylglutaric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxypropionic acid, methylcitrate, and lactic acid, which occur in the absence of multiple carboxylases. Cases with ketosis may excrete moderate amounts of 3-hydroxybutyric acid and acetoacetic acid and may have secondary dicarboxylic aciduria. Severe ketosis from any cause can result in a moderate increase in 3-hydroxyisovaleric acid (50 to 200 mmol/mole creatinine, normal is l to 20 mmol/mole creatinine), but usually less than that seen in this condition, and ketosis does not produce a secondary increase in 3-methylcrotonylglycine. The final diagnosis of simple 3-methylcrotonyl coenzyme A carboxylase deficiency and exclusion of multiple carboxylase deficiencies should be detected as reduced activity of this enzyme in the patient’s leukocytes and not associated with biotin treatment; or the enzyme activity should be reduced in fibroblasts under different biotin concentration culture conditions and other carboxylase activities should be normal. Because heterozyme activity is usually within the normal range, it cannot be detected by enzyme activity assay in leukocytes or cultured fibroblasts. Prenatal diagnosis can be made by measuring the increased concentration of 3-hydroxyisovaleric acid in amniotic fluid by stable isotope dilution and by measuring 3-methylcrotonyl coenzyme A carboxylase activity in chorionic villus specimens or cultured amniotic cells.