This is the first study performed to date that evaluates the genotype-phenotype relation in patients with a diagnosis of biotinidase enzyme deficiency with repeated measurements of biotinidase enzyme activity. Biotinidase enzyme activity is known to be affected by genetic and other factors, such as premature birth, neonatal jaundice, patient age and the temperature of the specimen during storage or transport, and so we believe that repeated measurements of plasma biotinidase activity facilitated a better evaluation of the diagnosis and disease severity in patients with suspected biotinidase enzyme deficiency. [4]
Previous study has reported an incidence of biotinidase enzyme deficiency of 1/7,116 according to the data published by the Ministry of Health. [1] Canda et al.’s [4] study of 259 patients with biotinidase enzyme deficiency reported partial and profound enzyme deficiency in 71.8% and 28.2% of the patients, respectively, while a different study of 203 patients reported partial enzyme deficiency and profound enzyme deficiency in 77% and 23% of the cases, respectively. [11] In the present study, the first enzymatic examination of the diagnosed with biotinidase enzyme deficiency group with homozygous or compound heterozygous BTD variants revealed partial and profound enzyme deficiency in 57.4% and 20.5% of cases, respectively, while repeated measurements revealed partial and profound enzyme deficiency in 60.2% and 15.4% of cases, respectively. Surprisingly, heterozygous enzyme deficiency was detected in 22.1% and 24.4% of the patients with homozygous or compound heterozygous mutations in the BTD gene in the first and repeated enzyme analyses, respectively. Furthermore, partial enzyme deficiency was detected in 57.1% and 64.2% of the cases with a heterozygous mutation in the BTD gene in the first and repeated enzyme analyses, respectively. In addition, we determined profound enzyme deficiency in one (0.6%) patient despite he had heterozygous mutation in BTD gene. As presented in Fig. 1, measuring enzyme activity once can also give falsely low results. These results raise the necessity of looking at more than one enzyme level. All these findings suggest that a definite diagnosis of biotinidase enzyme deficiency requires a molecular genetic investigation.
The ClinVar database lists 123 pathogenic and 83 likely pathogenic variants. [5] In the United States, according to a study, p.Gln436His (c.1308A > C), p.Ala151Thr (c.451G > A), p.Asp444His (c.1330G > C) and p.Asp232Gly (c.695A > G) mutations were found in 52% of newborns who had profound enzyme deficiency. [16] The two most common mutations detected in symptomatic patients with profound enzyme deficiency in the United States were c.98-104del7ins3 and p.Arg538Cys, [17] while the most common mutation in Europe was reported to be p.Asp444His. [18] In our country, p.Asp444His, p.Arg157His, c.98-104del7ins3 and p.Thr532Met were reported to be the most common mutations in the BTD gene. [10] While p.Arg157His (c.470G > A) was frequently detected in profound enzyme deficiency, p.Asp444His (c.1330G > C) was mostly detected in patients with partial enzyme deficiency. [10; 19] In those with profound enzyme deficiency, p.Arg157His (c.470G > A) was the most common, followed by c.98_104delinsTCC (p.C33Ffs*36). In a further study, 20 of 26 patients with homozygous p.Arg157His (c.470G > A) mutations were reported to have profound enzyme deficiency, among which p.Asp444His (c.1330G > C)/p.Arg157His (c.470G > A) compound heterozygous mutations were the most common in those with partial enzyme deficiency, followed by homozygous p.Asp444His (c.1330G > C). [4] Concurring with this study, the most common allele frequencies in the present study were p.Asp444His (c.1330G > C) (53.4%), p.Arg157His (c.470G > A) (15.7%) and c.98-104del7ins3 (TCC) frameshift (5.1%). Among the homozygous and compound heterozygous variants in the present study, p.Asp444His (c.1330G > C) homozygous, p.Asp444His (c.1330G > C)/p.Arg157His (c.470G > A) compound heterozygous and homozygous p.Arg157His (c.470G > A) were determined as the most common variants in our study.
Previous studies have found the p.Arg157His (c.470G > A), p.Cys186Tyr (c.557G > A), p.Thr532Met (c.1595C > T) and p.Gln456His (c.1368A > C) mutations to be associated with profound enzyme deficiency. [4] Deletions, insertions or nonsense mutations are often associated with a complete absence of biotinidase activity, and are common in symptomatic patients with biotinidase enzyme deficiency. [4; 7] Karaca et al. [8] reported frequent neurological findings (seizures, encephalopathy) in symptomatic patients with the homozygous c.98-104del7ins3 /p.C33Ffs*36 mutation. In the present study, biotinidase enzyme activity of all patients with the homozygous p.C33Ffs*36 and homozygous p.Thr176Arg (c.527C > G) mutations, and nearly 80% of patients with homozygous p.Arg157His (c.470G > A) mutation, were related with profound enzyme deficiency. Only 41.2% of the symptomatic patients had profound enzyme deficiency, and 11.8% of these had heterozygous enzyme deficiency. These findings show that there is a genotype-phenotype relationship in patients with biotinidase enzyme deficiency, while there is no correlation between biotinidase enzyme activity and the clinical severity of the disease.
The p.Asp444His (c.1330G > C) mutation is generally considered a mild variant in literature, and most compound heterozygous patients with p.Asp444His (c.1330G > C) simultaneously with a severe variant (causing profound enzyme deficiency) have a less severe enzyme deficiency (partial enzyme deficiency). There are reported homozygous cases with normal activity and partial biotinidase enzyme deficiency activity, among which, those with p.Asp444His (c.1330G > C) homozygous variants are expected to show activity similar to heterozygous for severe variants (approximately 50%). Approximately 36–49% of normal biotinidase activity in this allele is expected to account for 2–30% of normal biotinidase activity in the heterozygous p.Asp444H allele (wild type/ p.Asp444H). [3] We consistently identified a close relationship between biotinidase enzyme activity and p.Asp444His (c.1330G > C) mutation.
Wolf et al. [20] reported that those with homozygous p.Asp444His (c.1330G > C) could be expected to have 45–50% of the average normal serum biotinidase enzyme activity, and so biotin treatment is not required. However, cases have been reported in literature with the homozygous p.Asp444His (c.1330G > C) mutation and profound enzyme deficiency. [4; 12] Canda et al. reported severe diaper dermatitis responsive to biotin therapy in 10 patients with homozygous p.Asp444His (c.1330G > C) mutations, despite biotinidase enzyme activity of 40%, [4] while walking difficulties and hypotonia were reported in two patients with homozygous p.Asp444His mutations. [10] In the present study, biotinidase enzyme activity was analyzed 130 times in 28 patients with the homozygous p.Asp444His (c.1330G > C) mutation in the BTD gene, and while no significant differences were noted in terms of enzyme deficiency severity in the first and repeated measurements, heterozygous enzyme deficiency was detected in 43% of patients with the homozygous p.Asp444His (c.1330G > C) mutation. On the other hand, partial enzyme deficiency was identified in 29.7% of the patients with the heterozygous p.Asp444His (c.1330G > C) mutation in the biotinidase enzyme activity analysis. These results suggest that a genetic investigation is necessary for a definite diagnosis of biotinidase enzyme deficiency.
Profound enzyme deficiency was identified in one (3.6%) patient with the homozygous p.Asp444His (c.1330G > C) mutation, and skin findings (7.1%), hair findings (3.6%), speech problems (14.3%) and developmental delay (3.6%) were recorded in five patients with homozygous p.Asp444His (c.1330G > C) mutations. These findings suggest a need to revisit the claim that “biotin treatment is not required in patients with homozygous p.Asp444His (c.1330G > C)” made in previous publication. [21]
To conclude, this study has revealed that molecular genetic analysis is necessary for a definitive diagnosis of biotinidase enzyme deficiency, as biotinidase enzyme activity alone is inadequate for diagnosis and for the evaluation of disease severity. We believe that repeated measurements of biotinidase enzyme activity led us to better understand the genotype-phenotype relationship in patients with biotinidase enzyme deficiency.