Inherited multiple carboxylase deficiency is a rare autosomal recessive disorder, firstly described in 1971 as biotin-responsive β-methylcrotonylglycinuria (Br med et al., 1945). It was later characterized as a deficiency of four carboxylases: Pyruvate carboxylase (EC 6.4.1.1); Acetyl-CoA carboxylase (EC 6.4.1.2); Propionyl-CoA carboxylase (EC 6.4.1.3) and Methylcrotonyl-CoA carboxylase (EC 6.4.1.4). Biotin is an essential vitamin obtained from diet and from a recycling mechanism. Biotin acts as a coenzyme for the four carboxylation enzymes involved in gluconeogenesis, fatty acid synthesis, and amino acid catabolism.

Two inherited defects have been identified that affect the function of biotin (Sweetman, 1981): Biotinidase and Holocarboxylase synthase deficiency (HCS). Biotidinase deficiency lead to biotin depletion due to an inability to recycle endogenous biotin and to use protein bound biotin from diet (Wolf et al., 1983). In HCS deficiency, biotinylation of the four apocarboxylases is impaired (Burri et al., 1981).

HCS deficiency is a rare metabolic disorder. Newborn screening data allow to estimate an incidence at birth ranging from 1/150 000 (Vilarinho et al., 2010) to 1/300 000 (Niu et al., 2010). The typical presentation is usually a neonatal onset within the first days of life and include lethargy, hypotonia vomiting, seizures and severe metabolic acidosis with hyperlactatemia, ketosis and hyperammonaemia. This metabolic disorder leads to coma and death without biotin supplementation. Patients with less severe defect also present dermatologic signs such as rash, eczema, alopecia and develop psychomotor retardation.

We report the cases of two patients: an 11-year-old girl, referred to as “the first patient,” diagnosed after her first episode of decompensation with typical clinical manifestations, and her older brother, referred to as “the second patient,” diagnosed at 23 years-old. To our knowledge, these represent the latest symptomatic onsets in published cases of HCS deficiency.

The first patient is the fourth child of consanguineous parents. She was urgently admitted at 11 years-old to the pediatric emergency department due to lethargy and abdominal pain following 2 days of vomiting. Her past medical history was unremarkable with a normal psychomotor development and no difficulties in school.

On admission, clinical examination revealed a compensated hypovolemia with sinus tachycardia, hypotension, superficial polypnea and cutaneous signs of extracellular dehydration. Consciousness varied, with the Glasgow Coma Scale score ranging between 11 and 15. The patient was somnolent and had no fever. Skin examination was unremarkable and she had no alopecia. Initial investigations revealed a severe metabolic acidosis with ketosis (pH 7.01 and bicarbonate of 3 mmol/L). The biochemical examination showed the following: Hyperlactatemia (8.3 mmol/L), severe hypokalemia (2.18 mmol/L), creatinemia (164 μmol/L) and uremia (12 mmol/L) indicating an acute renal failure. Blood glucose concentration was 1.26 g/L, blood ammonia 28 μmol/L, liver transaminases were normal, with aspartate aminotransferase at 25 U/L and alanine aminotransferase at 15 U/L.

After rehydration and electrolyte correction, the clinical condition improved rapidly and the acidosis resolved almost completely. Lactate decreased to 2.7 mmol/L in few hours.

According to the clinical and biological picture, a presumptive diagnosis of a metabolic inherited disease was suspected.

Urinary organic acid analysis was suggestive for profile of an inborn disorder of biotin metabolism with very elevated levels of 3-hydroxyisovaleric acid, 3-hydroxypropionic acid, 3-methylcrotonylglycine and traces of methylcitric acid and tiglylglycine (Figure 1A). These markers were associated with huge peaks of lactate and ketone bodies. Acylcarnitine profile was in agreement with the urinary organic acid profile and showed increased values of 3-hydroxyisovalerylcarnitine (C5OH), propionylcarnitine (C3), tiglylcarnitine and/or 3-methylcrotonylcarnitine (C5:1), acetylcarnitine (C2) and 3-hydroxybutyrylcarnitine (C4OH). The normal serum biotinidase activity (149 nkat/L, within the normal range of 90–210 nkat/L) rules out biotinidase deficiency and suggests a diagnosis of HCS deficiency.

48 h after admission, biotin therapy was started with a dose of 20 mg/day and oral levocarnyl at 100 mg/kg per day.

The diagnosis was later confirmed by genetic analysis which revealed a novel variant on the HLCS gene at an homozygous state [NM_000411.8:c.995A>G; p. (Gln332Arg)] that have not been previously reported in the literature. Both parents were tested and found to be carriers of the allele in a heterozygous state. This very rare variant [one heterozygous carrier on gnomAD v2 and one in gnomAD v3 (Chen et al., 2022)] is predicted by spliceAI (Jaganathan et al., 2019; de Sainte Agathe et al., 2023) tool to partially abolish the canonical donor splice site of exon 5 and reinforce a cryptic site located 127 bp upstream (Figure 2A). For the mutated allele, the canonical and cryptic splice sites have a very close probability of being used for the splice, which would lead to one normal mRNA and a shorter one containing a premature termination codon. A previously published variant in the same codon was described in a case of HCS deficiency with a similar predicted effect on the splicing mechanism (Quinonez et al., 2017). Regarding the ACMG classification, the variant was classified “likely pathogenic” (Figure 2B).

The patient was discharged from the hospital after 7 days. After 3 years of metabolic follow-up, metabolic parameters normalized with no further episodes of decompensation, except for a 1-day hospitalization due to an intercurrent infection and vomiting, which resolved rapidly.

The second patient is one of her older brother. His medical history mentions childhood epilepsy, for which he was treated with Valproate until he was 5 years old, a mild global developmental delay, a behavior disorder temporarily treated with neuroleptic and an episodic decrease in visual acuity. No acute episodes were documented. In light of the family history, metabolic investigations were initiated when he was 23 years old, following the diagnosis of his sister. Urinary organic acid (Figure 1B) analysis showed peaks of lactic acid, 3-hydroxybutyric acid and specific markers of a biotin metabolism disorder: 3-hydroxyisovaleric acid, 3-hydroxypropionic acid, 3-methylcrotonylglycine, methylcitric acid and tiglylglycine. These peaks were in smaller proportion than in his sister’s profile confirming that his sample was collecting outside any metabolic decompensation. As his sister, biotinidase activity was normal (186 nkat/L). No acylcarnitine profile or genetic analysis could be performed as the patient was unavailable for these tests. Biotin therapy was started with a dose of 100 mg/day for 1 month then 10 mg/day and oral levocarnyl at 3g/day.

The other two siblings did not exhibit any symptoms and were not tested.

The cases of two siblings with HCS deficiency have been described, including an eleven-year-old girl who presented with a pattern of decompensation associated with metabolic acidosis, which rapidly resolved with symptomatic management. The late diagnosis of HCS deficiency was confirmed and the patient was managed with oral biotin, without any episodic decompensation 3 years after the diagnosis. The family history revealed no prior instances of metabolic decompensation, such as episodes of metabolic acidosis or related symptoms, before this case. An older brother was then diagnosed through family screening, but he presented an history of non-metabolic clinical symptoms such as epilepsy and mild developmental delay. Interestingly, HCS with isolated epilepsy have already been described (Donti et al., 2016; Suormala et al., 1997).

The variant found was not reported in the literature, but a previously published case from Quinonez et al. (2017) reported neonatal HCS deficiency with a close variant (c.996G>C) abolishing the same splice site at the end of exon 5. However, the clinical impacts of these two cases are difficult to compare since the complete genotype was different: our case is homozygous for the splice variant and the Quinonez et al. (2017) case was in compound heterozygosity for c.996G>C variant and an inversion of chromosome 21 which disrupt the HLCS gene.

To investigate an effect of genotype on the age of onset of HCS deficiency, we performed a mini-review of cases at two extremes of age at which the first symptoms of HCS deficiency developed: before 1 month and after 1 year.

The review, conducted in July 2024, identified a total of 20 cases with initial symptoms appearing before 1 month, and 9 cases with symptoms emerging after 1 year. Table 1 summarizes the data from this mini-review.

The genotypes between the early and late onset groups are non-concordant, which indicates that the genotypes potentially influence the timing of the condition. The variant p. (Arg508Trp) is an exception, as it was found in both a late onset case (in a homozygous state) and an early onset case (in a compound heterozygous state with a loss-of-function variant).

Some variants are recurrent and are associated either with late cases (variants with partial splicing effect for c.995A>G and c.1519 + 5G>A) or either with early cases [p. (Leu216Arg) and p. (Leu237Pro)]. The other variants appear to be private. Notably, all missense variants excepted one show elevated levels of pathogenic prediction according to the REVEL algorithm (>0,75) (Ioannidis et al., 2016).

It appears feasible to assess phenotype severity through genotype analysis if the aforementioned recurrent variants are detected in the homozygous state. Thus splice variants could be associated with late forms, the p. (Leu216Arg) (Slavin et al., 2014) and p. (Leu237Pro) (Aoki et al., 1995) variants with severe early onset forms. The p. (Arg508Trp) variant is relatively common in late-onset cases, but a publication by Ling et al. (2023) has also demonstrated its frequency in intermediate-onset cases (those occurring between 1 month and 1 year) so we cannot conclude on its severity.

Interestingly, the HLCS gene is included in ongoing studies for genetic newborn screening (Holm et al., 2018). This approach can prevent symptoms and complications at early stage, as well as identify late-onset cases at risk of decompensation. A better understanding of the genotype-phenotype correlation would be beneficial, as it would allow for the anticipation of the early or late onset of the condition in the context of genetic newborn screening.

Additionally, prenatal biotin administration may improve the condition in severe early cases if a previous child in the family is known to have an HCS defect (Suormala et al., 1998).

We presented two cases of holocarboxylase synthase deficiency in a family, both of which were unusually diagnosed at a late stage. The first patient’s outcome was favorable following acute treatment and long-term biotin administration; the second patient did not present specific metabolic signs at 23 years old but a mild developmental delay. Those cases underscore the importance of conducting standard metabolic biochemical assessments when faced with suspicious clinical presentations, even in pre-adolescent children and adults. An analysis of genotypes among cases reported in the scientific literature reveals that the nature of the variants may only partially account for the delayed diagnosis in some patients.