Multiple carboxylase deficiency (biotinidase deficiency and holocarboxylase synthetase deficiency): clinical guidelines

List of Abbreviations

C5OH — 3-hydroxyisovalerylcarnitine;

ACC — acetyl-CoA carboxylase;

MCC — β-methylcrotonyl-CoA carboxylase;

MCD — multiple carboxylase deficiency;

BTD — biotinidase deficiency;

HLCS — holocarboxylase synthetase deficiency;

PC — pyruvate carboxylase;

PCC — propionyl-CoA carboxylase;

MS/MS — tandem mass spectrometry.

Terms and Definitions

Metabolic crisis is a critical, life-threatening condition triggered by adverse factors that intensify cellular catabolism with the accumulation of toxic derivatives, presenting with acute encephalopathy, recurrent vomiting, and seizures.

Neonatal screening involves mass screening of newborns for the most common congenital and hereditary disorders to prevent severe forms of the diseases before the development of clinical symptoms.

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Multiple carboxylase deficiency (MCD) is caused by two inherited disorders of biotin metabolism: biotinidase deficiency (BTD) and holocarboxylase synthetase deficiency (HLCS). Reduced activity of these enzymes leads to a deficiency of four biotin-dependent carboxylases. Both disorders share clinical and biochemical similarities. The mainstay of treatment is high-dose biotin therapy. When biotin therapy is initiated in a timely manner, the prognosis for both disorders is favorable. However, delayed initiation of therapy can lead to irreversible neurological damage.

Biotinidase deficiency (BD) (OMIM 253260) is an autosomal recessive disorder caused by mutations in the BTD gene, which encodes the biotinidase enzyme. Enzyme dysfunction disrupts the cleavage of biotin from proteins and, consequently, impairs the function of all biotin-dependent carboxylases. The BTD gene is mapped to the short arm of chromosome 3 (3p25).

Holocarboxylase synthetase deficiency (HLCSD) (OMIM 253270) is an autosomal recessive disorder caused by mutations in the HLCS gene, which encodes the holocarboxylase synthetase enzyme, leading to impaired binding of biotin to biotin-dependent carboxylases. The disorder is similar in its manifestations to BD, but is characterized by an earlier onset and a more severe clinical course. The holocarboxylase synthetase gene, HLCS, is mapped to the long arm of chromosome 21 (21q22).

1.2 Etiology and pathogenesis of the disease or condition (group of diseases or conditions)

Biotin (vitamin H or B7) is a water-soluble vitamin belonging to the B-complex. Biotin enters the body via dietary intake and, potentially, through the synthetic activity of the gastrointestinal microflora. The biotin molecule consists of a heterocyclic ring with carboxyl groups attached to its aliphatic side. Biotin serves as a coenzyme for four carboxylases: pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), β-methylcrotonyl-CoA carboxylase (MCC), and acetyl-CoA carboxylase (ACC). These enzymes participate in critical intermediate cellular metabolic reactions and play a major role in gluconeogenesis, fatty acid synthesis, and amino acid catabolism [1]. Specifically, PC catalyzes the carboxylation of pyruvate to oxaloacetate (a key step in gluconeogenesis, lipogenesis, and neurotransmitter biosynthesis); ACC catalyzes the first step of fatty acid biosynthesis (the carboxylation of acetyl-CoA to malonyl-CoA); PCC catalyzes the carboxylation of propionyl-CoA in the metabolism of branched-chain amino acids and certain fatty acids; MCC catalyzes the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA in the metabolism of leucine and isovaleric acid [2]. ACC functions primarily in the cytoplasm, whereas the other four holocarboxylases function in the mitochondria and on the outer mitochondrial membrane.

Each of the carboxylases for which biotin serves as a covalently bound cofactor is synthesized as an inactive apoenzyme, which is subsequently biotinylated via two partial reactions catalyzed by the holocarboxylase synthetase enzyme. Biotin-containing degradation products—biocytin (ɛ-N-biotinyl-L-lysine) and biotinyl peptides—are acted upon by biotinidase, which cleaves the amide bond between lysine and biotin [3]. The released biotin is recycled and enters the free biotin pool (Fig. 1).

Impaired carboxylase function leads to the accumulation of intermediate substrates of the enzymatic reactions controlled by carboxylases. These substrates and their derivatives exert toxic effects on the central nervous system and other tissues, resulting in metabolic acidosis and secondary hyperammonemia.

There are two primary defects in the human biotin recycling cycle: biotinidase deficiency (BD) and holocarboxylase synthetase deficiency (HLCSD). Both disorders lead to MCD. HLCSD (OMIM 253270), also known as early (neonatal) MCD, is a disorder of biotinylation. BD (OMIM 253260), also known as late (juvenile) MCD, is a disorder of biotin recycling. Isolated deficiencies of individual carboxylases are rare.

Biotinidase is a 543-amino acid protein with a molecular weight of 85 kDa [3]. Its encoding gene, BTD, is localized on the short arm of chromosome 3 (3p25) and consists of 4 exons and 3 introns. The total length of the coding sequence is 1,629 base pairs [4]. To date, at least 165 pathogenic mutations in BTD are known [5]. The most frequent mutations are a 7-base pair deletion with a 3-base pair insertion, c.98_104delGCGGCTGinsTCC (p.Cys33Phefs*36), which is found in 60% of patients with BD [6], and the missense mutation c.1612C > T (p.Arg538Cys) [7]. Both mutations in the homozygous or compound heterozygous state lead to profound BD. Also quite common are the missense variant c.1368A > C (p.Gln456His; Q456H) and the variant c.1330G > C (p.Asp444His; D444H), which lead to profound BD [8]. The compound heterozygote c.1330G > C; c.511G > A (p.D444H;A171T) reduces biotinidase activity by 52% and, together with the p.Cys33Phefs*36, p.Gln456His, and p.Arg538Cys variants, accounts for 65% of all BD cases [9, 10].

Free biotin, primarily derived from diet, is actively transported across the intestinal membrane and the blood-brain barrier. This free biotin pool supplies various apocarboxylases (pyruvate carboxylase [PC], acetyl-CoA carboxylase [ACC], propionyl-CoA carboxylase [PCC], and beta-methylcrotonyl-CoA carboxylase [MCC]). Holocarboxylase synthetase assembles active holocarboxylases by covalently attaching biotin to their active sites. Holocarboxylases are essential for gluconeogenesis, fatty acid synthesis, and the catabolism of several branched-chain amino acids. Subsequently, holocarboxylases are proteolytically cleaved into biocytin or small biotinyl peptides. These compounds are cleaved by biotinidase to yield lysine and free biotin, which can enter the free biotin pool, thereby recycling the vitamin. ATP = adenosine triphosphate.

Holocarboxylase synthetase is a 726-amino acid protein with a molecular weight of 81 kDa. Its encoding gene, HLCS , is localized on the long arm of chromosome 21 (21q22) and consists of 11 exons [1]. The total length of the coding sequence is 2,400 base pairs. At least 35 pathogenic variants of the HLCS gene are known in the literature [1]. One of the frequent variants is c.1522C > T (p.Arg508Trp; p.R508W), detected in Thailand, China, France, Iran, and the USA [11]. In Japan, the prevalent pathogenic variants are c.710T>C (p.Leu237Pro) and c.780delG (p.G261VfsX20) [12], whereas in China, c.1088T > A (V363D) predominates [13].

It has also been shown that lysine residues of histones H3 and H4 are biotinylated by holocarboxylase synthetase (in vivo) and biotinidase (in vitro). Biotinylated histones are likely involved in important biological functions such as gene silencing, chromatin condensation, DNA repair, and cell proliferation [14]. However, according to studies, the amount of biotinylated histones in vivo is < 0.001%, which casts doubt on their physiological significance [15].

1.3 Epidemiology of the Disease or Condition

BD is a panethnic disorder. The prevalence of BD ranges from 1:40,000 to 1:60,000 newborns worldwide [16]. In some countries, such as Turkey and Saudi Arabia, the prevalence is higher due to the high frequency of consanguineous marriages. Based on newborn screening results, the incidence of profound BD in the USA is 1:80,000 newborns, and partial BD ranges from 1:31,000 to 1:40,000 [17]. The global incidence of HLCSD is 1:200,000 live births [18]. The incidence of BD and HLCSD in the Russian Federation has not been established.

1.4 Features of Coding the Disease or Condition

According to ICD-10, BD and HLCSD belong to Class IV — Endocrine, nutritional and metabolic diseases.

ICD-10: E53.8 — Deficiency of other specified B-group vitamins.

ICD-11: 5C50.E0 — Classical organic acidurias.

OMIM: 253260 (BD) and 253270 (HLCSD).

ORPHA code: 79241 (BD) and 79242 (HLCSD).

1.5 Classification of the disease or condition

Multiple carboxylase deficiencies are divided into early- and late-onset forms. Early-onset includes MCD, while late-onset includes BTD. BTD, in turn, depending on biotinidase enzyme activity, is divided into profound (< 10% of normal serum biotinidase activity) and partial (10–30% of normal). Normally, serum biotinidase activity ranges from 4.4 to 12 nmol/min/mL [19].

1.6 Clinical presentation of the disease or condition

Biotinidase deficiency (BTD)

In early childhood, the most frequent initial symptoms of BTD are epileptic seizures: in the majority of cases, myoclonic seizures are observed, and infantile spasms are not uncommon [20–22]. Sometimes, muscular hypotonia is the main clinical symptom [21]. In a number of cases, BTD manifests with psychomotor development delay, breathing pattern disorders (Kussmaul breathing, laryngeal stridor, apnea), seborrhea, atopic dermatitis, alopecia areata and/or totalis, and persistent conjunctivitis [3, 23]. Most patients have a combination of neurological and cutaneous clinical manifestations. Frequent neurological disorders include sensorineural hearing loss and vision impairment (most often due to optic atrophy) [24–26]. Sudden infant death syndrome associated with BTD has been reported [27].

Profound BTD most frequently manifests between 3 and 5.5 months of age. In rare cases, the disease debuts during adolescence or in the first week of life. The severity of clinical manifestations depends on the level of biotinidase activity [28]. Thus, high residual enzyme activity (30 to 25% of normal) is characteristic of benign juvenile forms of BTD. When the disease debuts in adolescence, the initial symptoms are muscle weakness, spastic paraparesis, and visual disturbances [29]. Later, sudden vision loss with progressive optic neuropathy may develop [30, 31].

Biotinidase activity in the human brain is extremely low; therefore, for normal neuronal function, a sufficient and constant supply of biotin across the blood-brain barrier is required. A decrease in biotin concentration in BTD at the early stages of the disease leads to reduced PC activity, which causes the accumulation of lactate (lactic acid) in the brain [32]. This local lactic acidosis primarily causes neurological symptoms, while other signs of the disease may manifest later. Ketoacidosis is a sign of prolonged biotin deficiency in the body and may not be detected at the initial stages of the disease. It is believed that the cause of sensorineural hearing loss is the accumulation of organic acids, biocytin, and larger biotinyl proteins [32, 33]. The decrease in protective fatty acids levels (resulting from impaired PCC function) is probably the cause of alopecia and skin rash [34].

The toxic effect explains morphological changes in the cerebellum (absence of Purkinje cells, rarefaction of the granular cell layer and proliferation of the Bergmann glial layer, gliosis of the white matter and dentate nuclei) and the cerebrum (necrosis with vascular proliferation and macrophage infiltration in the perivascular space) [35]. Changes in subcortical structures and the spinal cord, similar to those in Leigh disease, have been described [36]. Myelin fibers are affected to a greater extent than neuronal cell bodies; therefore, impaired brain myelination processes are frequently described in BTD.

Cases of disease debut with pyramidal symptoms and the development of progressive spastic paresis due to spinal cord myelopathy, the severity of which decreased with biotin therapy, have been described in the literature [37, 38]. Studies show that long-term biotin administration prevents the development of spastic paraparesis and sensorineural hearing loss in patients with BTD [38, 39]. Newborns with BTD diagnosed before the onset of clinical symptoms (e.g., via neonatal screening) and receiving high-dose biotin treatment have normal development [25].

Cases of asymptomatic course of profound BTD are known [17, 40–42].

Patients with partial BTD have a mild form of the disease. Symptoms usually develop under stress: due to surgery, during infection, prolonged fasting, etc. The literature describes a case where a child with partial BTD who did not receive biotin treatment experienced hypotonia, skin rash, and hair loss during an episode of gastroenteritis at six months of age. Symptoms resolved with biotin treatment [43].

The genotype-phenotype correlation of BTD has not been fully established. Deletions, insertions, or nonsense nucleotide variants usually lead to a complete loss of biotinidase enzymatic activity, whereas missense variants may or may not lead to its complete loss. In one study, children with symptoms of profound BTD carrying null mutations had a greater likelihood of developing hearing loss than children with missense mutations [44]. Some genotypes correlate with profound BTD, while others correlate with partial BTD. Most pathogenic variants of the BTD gene cause a complete or almost complete loss of biotinidase enzyme activity. The combination of two such alleles (homozygosity or compound heterozygosity) leads to profound BTD. For example, compound heterozygosity for p.Asp444His and a pathogenic variant leading to profound BTD will result in approximately 20–25% of normal serum enzymatic biotinidase activity [45].

Holocarboxylase synthetase deficiency (MCD)

MCD is known as the early (neonatal) form of multiple carboxylase deficiency. The disease typically manifests in the first few hours, days, or weeks of life, less commonly in the first year of life or childhood. The clinical manifestations of MCD are very similar to BTD. The initial clinical symptoms are refusal to feed, recurrent vomiting, increased drowsiness or irritability, respiratory disorders, muscular hypotonia, and exfoliative dermatitis. Some children also exhibit skin rash, alopecia, developmental delay, metabolic acidosis, hyperammonemia, and, as in BTD, altered urinary organic acid profiles characterized by elevated concentrations of β-hydroxyisovalerate, β-methylcrotonylglycine, β-hydroxypropionate, methylcitrate, C5OH, lactate (lactic acid), and tiglylglycine [46]. Occasionally, patients with MCD experience hypoglycemia as a consequence of reduced PC activity [47, 48].

As with BTD, symptom severity decreases with biotin administration.

In the absence of treatment, the disease progresses rapidly, developing resistant epileptic seizures, metabolic acidosis, cerebral edema, depressed consciousness, and coma [46]. Such patients frequently exhibit subependymal cysts, ventricular enlargement, and intraventricular hemorrhage [49].

Since MCD and BTD are biochemically and clinically similar, molecular genetic analysis of the BTD and HLCS genes is necessary to establish a precise diagnosis.

2. Diagnostics, medical indications and contraindications for diagnostic methods

Please note that in accordance with the requirements for the development of clinical guidelines, each recommendation statement must indicate the strength of recommendations and the evidence base in accordance with the grading scales for the levels of evidentiary certainty (LEC) and the levels of recommendation persuasiveness (LRP). For many statements, the LRP and LEC will be low due to the lack of high-design clinical trials dedicated to them. Despite this, they are necessary elements of patient examination to establish a diagnosis and choose treatment tactics.

Criteria for establishing the diagnosis and conditions

The diagnostic criterion is the detection of decreased blood biotinidase enzyme activity, elevated concentrations of β-hydroxyisovalerate, β-methylcrotonylglycine, β-hydroxypropionate, methylcitrate, lactate, and tiglylglycine upon quantitative and qualitative analysis of urinary organic acids, as well as elevated blood acylcarnitine concentrations (C5OH) [46]. It should be noted that hydroxyisovalerylcarnitine (C5OH) is not elevated in the blood during the neonatal period in the majority of patients.

Identification of pathogenic variants in the BTD and HLCS genes via DNA diagnostics confirms the diagnosis at the molecular genetic level.

Differential diagnosis is carried out with inherited organic acid metabolism disorders, mitochondrial diseases, as well as meningitis, primary immunodeficiency, acrodermatitis enteropathica, sensorineural hearing loss, autism, myelopathy, optic atrophy, seborrheic dermatitis, infantile spasms [50], and neuromyelitis optica spectrum disorders [51].

2.1 Complaints and Anamnesis

When taking the medical history and collecting complaints, attention should be paid to the following complaints and anamnestical events:

− burdened family history (similar symptoms in siblings of the proband, consanguineous marriage);

− disease manifestation before 3–6 months of age;

− feeding disorders;

− unexplained restlessness, crying, limb tremor, hyperexcitability;

− lethargy, weakness, fatigue;

− respiratory disorders, including hyperventilation, laryngeal stridor, and apnea episodes;

− altered level of consciousness ranging from drowsiness to coma;

− epileptic seizures resistant to antiepileptic therapy;

− hearing loss of the sensorineural type;

− vision loss (partial optic atrophy, neuromyelitis optica);

− alopecia (total or partial);

− atopic dermatitis;

− recurrent conjunctivitis;

− undulating (crisis-like) course of the disease;

− cessation/delay/loss of previously acquired psychomotor skills;

− diffuse muscular hypotonia.

In childhood, the following symptoms are also observed:

− gait disturbance (spastic paraparesis, cerebellar ataxia, polyneuropathy);

− progressive visual impairment (partial optic atrophy, neuromyelitis optica);

− hearing loss (sensorineural hearing loss);

− encephalomyelopolyradiculoneuropathy;

− delayed/impaired psycholinguistic and speech development;

− alopecia;

− fluctuating (crisis-like) course of the disease.

Children or adults with untreated partial biotinidase deficiency may present with any of the above signs and symptoms, but manifestations are mild and occur only during stress, intercurrent infections, or surgical interventions.

Complaints and anamnesis are also described in the "Clinical Presentation" section.

2.2 Physical Examination

During the examination, attention should be paid to the main clinical manifestations of BD and HCD:

− total or partial alopecia;

− atopic dermatitis;

− diffuse muscular hypotonia ("floppy infant syndrome");

− delayed psychomotor development;

− refractory epileptic seizures;

− hepatosplenomegaly (in 10% of cases).

2.3 Laboratory Diagnostics

The main laboratory methods for confirming the diagnosis of BD and HCD include determining biotinidase enzyme activity in plasma or serum, quantitative and qualitative analysis of urinary organic acids, blood acylcarnitine testing, and molecular genetic testing of the BTD and HLCS genes. These studies are performed in specialized genetic laboratories.

Assessing the level/activity of biotinidase in the blood is one of the primary confirmatory biochemical tests for BD, allowing differential diagnosis with other inherited metabolic disorders [52]. Normally, serum biotinidase activity is 4.4–12 nmol/min/mL [19]. Typically, testing biotinidase activity requires 1–2 mL of serum or plasma. Samples must be frozen immediately and kept frozen until testing. Blood spots collected for newborn screening must be thoroughly dried before shipping to the laboratory, as high humidity leads to significant loss of enzyme activity. Reference ranges should be established by each laboratory. It is important to consider the patient's age, as biotinidase activity in term newborns is 38–70% of the mean normal adult biotinidase activity [53]. Newborn biotinidase activity usually increases during the first days or weeks of life [54].

Comments: neonatal screening involves measuring biotinidase enzyme activity in dried blood spots. Approximately 50% of false-positive results are related to prematurity, and most others are due to pre-analytical errors (improper storage and transport conditions). The determination of biotinidase activity allows for the detection of both profound (total) biotinidase deficiency (< 10% of mean normal activity) and partial deficiency (10–30% of mean normal activity). Heterozygous carriers of the disease may also exhibit reduced enzyme activity (approximately 50% of normal) [79].

Comments: for BD at the preclinical stage, C5OH concentrations may be within the normal range. The screening algorithm for BD and HCD is given in Appendix B.

Comments: characteristic of BD and HCD is an elevated concentration of β-hydroxyisovalerate, β-methylcrotonylglycine, β-hydroxypropionate, methylcitrate, lactate, and tiglylglycine [17, 46].

Comments: normally, serum biotinidase activity is 4.4–12 nmol/min/mL [19]. Newborn biotinidase activity usually increases during the first days or weeks of life [54].

Comments: most pathogenic variants in patients with BD can be detected by Sanger sequencing of all exons and adjacent intron regions of the BTD gene, but in a small percentage of cases, nucleotide substitutions cannot be detected using standard methods. The following frequent pathogenic variants have been described: c.98_104delGCGGCTGinsTCC (p.Cys33Phefs*36), which is found in approximately 60% of patients on at least one allele [6], and the c.1612C > T (p.Arg538Cys) variant [7]. Both variants lead to total BD. Almost all patients with partial BD have the c.1330G > C (p.Asp444His; D444H) variant on one allele combined with a pathogenic variant associated with total BD [57]. Variants c.1368A > C (p.Gln456His) and c.1330G > C (p.Asp444His), which lead to total BD, are also detected quite frequently [8]. The c.1330G > C; c.511G > A (p. D444H; A171T) compound heterozygote reduces biotinidase activity by 52% and, together with the p.Cys33Phefs*36, p.Gln456His, and p.Arg538Cys variants, accounts for 65% of all BD cases [9, 10]. An initial and follow-up appointment (examination, consultation) with a medical geneticist is required to determine the management strategy and subsequent interpretation of molecular genetic test results [50, 72, 74].

Comments: one of the frequent variants is c.1522C > T (p.Arg508Trp; p.R508W), detected in Thailand, China, France, Iran, and the USA [11]. In Japan, the pathogenic variants c.710T4C (p.Leu237Pro) and c.780delG (p.G261VfsX20) predominate [12], while in China, c.1088T > A (V363D) is most common [13]. An initial and follow-up appointment (examination, consultation) with a medical geneticist is required to determine the management strategy and subsequent interpretation of molecular genetic test results [50, 72, 74].

Comments: the study is performed during initial diagnosis and dynamic follow-up.

Comments: laboratory precursors of a metabolic crisis include a tendency toward metabolic acidosis with base deficit.

Comments: the frequency of testing depends on the patient's condition.

2.4 Instrumental Diagnostics

Comments: brain CT/MRI in patients with inherited metabolic diseases reveals frontotemporal atrophy, ventricular enlargement, delayed myelination, subdural fluid collections, subependymal cysts, and intraventricular hemorrhage [17, 49, 63]. It should be noted that these abnormalities are not highly specific and may be observed in other conditions. General anesthesia (Anesthesiological management, including early postoperative care) is performed according to indications.

Comments: BD and MCD are characterized by epileptic seizures resistant to antiepileptic therapy.

Comments: patients with BD and MCD present with sensorineural hearing loss [24, 39, 44, 64]. Studies are performed depending on the clinical situation.

2.5 Other Procedures

2.5.1 Specialist Consultations and Monitoring

Managing patients with a confirmed diagnosis of BD and MCD requires a multidisciplinary approach because these diseases are characterized by multiple organ and system involvement, necessitating coordinated care by specialists from various fields. Initial and follow-up appointments (examinations, consultations) are indicated with a medical geneticist, ophthalmologist, neurologist, otolaryngologist-audiologist, pediatrician/general practitioner/family physician, dermatovenereologist, as well as other specialists for patients with BD and MCD who have dysfunctions of the respective organs and systems.

Comments: when the disease debuts in adolescence, visual disturbances (decreased visual acuity, scotomas, optic atrophy) are among the first symptoms of BD and MCD [24–26, 29–31]. Sudden vision loss with progressive optic neuropathy may develop later [30, 31]. The examination is performed once every 6 months and as clinically indicated.

Comments: the medical geneticist performs differential diagnosis, determines the tactics for molecular genetic and biochemical diagnostics, and interprets the results of molecular genetic testing.

Comments: myoclonic seizures are observed in most cases at an early age, and infantile spasms are also common [20–22]. When the disease debuts in adolescence, the first symptoms are muscle weakness and spastic paraparesis [29]. In some cases, the disease manifests as psychomotor retardation. The Alberta Infant Motor Scale is provided in Appendix G1. Visit intervals should be determined individually, taking into account the patient's age and disease severity.

Comments: patients need testing using approved methods to assess intellectual development. Psychological support for patients may require a course of sessions depending on the individual characteristics of the patient and family.

Speech therapy services:

• Medical speech therapy assessment for dysphagia;

• Medical speech therapy assessment for aphasia;

• Medical speech therapy assessment for dysarthria;

• Medical speech therapy procedure for dysphagia;

• Medical speech therapy procedure for aphasia;

• Medical speech therapy procedure for dysarthria;

• Medical speech therapy tonal-rhythmic procedure;

• Medical speech therapy procedure using interactive information technologies.

2.5.2 Differential Diagnosis

Patients with clinical signs characteristic of BD and MCD require differential diagnosis with meningitis, primary immunodeficiency, acrodermatitis enteropathica, sensorineural hearing loss, autism, myelopathy, optic atrophy, seborrheic dermatitis, infantile spasms, and neuromyelitis optica spectrum disorders [50, 51].

The algorithm for differential diagnosis of inherited metabolic diseases with other disorders associated with altered C5OH levels is shown in Fig. 1B in Appendix B.

3. Treatment, including drug and non-drug therapies, diet therapy, and pain management

The main principles of therapy for BD and MCD aim to prevent metabolic decompensation (metabolic crisis), reduce the production of toxic organic acids, prevent toxic damage to the brain and internal organs, while ensuring normal growth and development in children.

The core components of treatment for patients with BD and MCD are high-dose biotin preparations or specialized clinical nutrition products containing biotin. Antiepileptic drugs and other types of symptomatic therapy are prescribed according to indications.

3.1 Pathogenetic treatment

Comments: all children with clinical manifestations of BD and MCD show improvement after treatment with #biotin at a dose of 5–20 mg daily. All patients identified through neonatal screening with biotinidase activity of less than 10% must receive biotin.

#Biotin is usually available in tablet form. To administer #biotin to an infant or young child, the tablet can be crushed and mixed with breast milk or formula in a spoon, medicine dispenser, or medical injection syringe. The tablet contents should not be placed in a bottle because the mixture will stick to the bottle and/or fail to pass through the nipple, leading to inconsistent dosing.

Therapy with #biotin normalizes metabolic parameters and controls epileptic seizures. Hair growth in children with alopecia resumes within a few weeks or months. Optic atrophy and hearing loss may persist, especially if treatment was initiated late against the background of severe neurological disorders. In children with partial biotinidase deficiency, symptoms may manifest during conditions associated with increased catabolism.

Comments: dosage according to the instructions. The amount of the resulting solution to be taken and the frequency of administration are prescribed by the attending physician depending on the stage and severity of the disease.

3.1.1 Management of patients during a metabolic crisis

Comments: in the event of a threat or development of a metabolic crisis, treatment must be initiated immediately. Therapeutic measures are aimed at stopping the production and accumulation of toxic organic compounds and eliminating them from the body. Treatment tactics for children during a crisis include correction of metabolic acidosis and water-electrolyte disturbances, as well as energy support. In the event of a threat or development of a metabolic crisis, emergency hospitalization is necessary for the immediate provision of intensive care and prevention of life-threatening complications.

Treatment of a metabolic crisis is carried out in the intensive care unit by an anesthesiologist-resuscitator, as well as other medical specialists (neurologist, nephrologist, gastroenterologist, etc.). Acid-base balance and electrolytes should be monitored to avoid hypernatremia and hypokalemia. In severe cases, peritoneal dialysis or mechanical ventilation may be required.

3.2 Symptomatic treatment

Symptomatic treatment is prescribed according to specialists' indications (see Section 5.1).

3.3 Surgical Treatment

3.4 Pain Management

Special pain management methods are not provided.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

Specific rehabilitation is not required for patients with IEM. The range of rehabilitation measures for patients with IEM may include sessions with a psychologist/neuropsychologist, speech therapist-defectologist, recovery in specialized sanatoriums, as well as social adaptation with the participation of specialists and social workers, and massage courses.

5. Prevention and Dispensary Observation

5.1 Prevention

Patients with BD and MCD should undergo routine preventive vaccination in accordance with the national immunization schedule, including vaccination against influenza and rotavirus infection, observing age restrictions for immunobiological drugs against the background of strict compliance with medical prescriptions in order to form an effective immune response and prevent the development of metabolic crises. Vaccination should preferably be carried out under the supervision of specialists experienced in working with patients with metabolic disorders. In the post-vaccination period, vaccinated individuals should be closely monitored with repeated body temperature measurements.

Patients with BD and MCD should also avoid consuming raw eggs, as they contain avidin — an egg white protein that binds biotin, thereby reducing its bioavailability [70].

Comments: as with other autosomal recessive diseases, in BD and MCD the risk of having an affected child for each pregnancy is 25%. Prenatal diagnosis of diseases that respond to effective therapy is possible, but ethically questionable. The decision to perform it should be made after a detailed discussion of all risks with the family. A follow-up visit may be necessary to discuss test results and determine further diagnostic tactics, if required.

Comments: at least once every 6 months.

Comments: at least once every 6 months.

Comments: at least once every 6 months.

5.2 Dispensary monitoring

Annually, patients with an established diagnosis of BD and MCD must undergo an advanced medical examination (dispensary check-up). A multidisciplinary approach is necessary for patient monitoring because these diseases are characterized by damage to multiple organs and systems, which in turn dictates the need for joint management of the patient by specialists of various profiles. To ensure comprehensive therapy and its timely correction if necessary, initial and follow-up consultations by a geneticist, ophthalmologist, neurologist, otorhinolaryngologist-audiologist, pediatrician/general practitioner/family physician, dermatovenereologist, medical rehabilitation physician, physical and rehabilitation medicine physician, as well as physicians of other specialties are indicated for patients with BD and MCD who have dysfunctions of the corresponding organs and systems.

Patients with symptomatic epilepsy require monitoring by a neurologist with experience in working with epilepsy patients.

Necessary rehabilitation measures are also carried out. Parents must be trained in the rules of organizing therapy during the interictal period and during the threat of a metabolic crisis. The child must always carry an information card indicating emergency procedures during the onset of a metabolic crisis.

Comments: intervals between visits should be determined individually, taking into account age, disease severity, metabolic stability, and adherence to dietary therapy.

Comments: at least once a year or more often if necessary.

Comments: at least once a year or more often if necessary.

Comments: at least once a year or more often if necessary.

Comments: at least once a year or more often if necessary.

Comments: the complete blood count is monitored at least once a year.

Comments: the routine clinical blood chemistry test is monitored at least once a year.

Comments: blood C5OH levels as a result of treatment should be within the normal range.

Comments: the frequency of testing depends on the patient's condition.

Comments: the frequency of testing depends on the patient's condition.

Comments: frequency of tests depends on the patient's condition.

6. Organization of Medical Care

Indications for scheduled hospitalization in a medical organization:

1. Provision of diagnosis and treatment requiring prolonged (daytime or round-the-clock stay) medical observation and monitoring of clinical and laboratory parameters.

2. Need for various types of expert evaluations or examinations in a medical organization when they cannot be performed on an outpatient basis (including issuing a conclusion of a federal concilium/medical commission).

Indications for emergency hospitalization in a medical organization:

1. Acute life-threatening conditions and conditions threatening the development of complications that require urgent treatment, including intensive care, as well as round-the-clock medical observation and special types of examination and treatment.

Indications for discharging a patient from a medical organization:

1. Absence of threat to the patient's life and threat of complications requiring urgent treatment upon completion of diagnostic measures.

2. Stabilization of the condition and main clinical and laboratory parameters of the pathological process, absence of the need for prolonged medical observation (daytime or round-the-clock stay).

3. The patient's examination and treatment plan has been completed, and recommendations for further monitoring and treatment have been given.

4. Need to transfer the patient to another medical institution or social security institution.

7. Additional Information

The prognosis of the patient's condition and mental development level depends on the severity of the disease, as well as the timing of initiation of specialized therapy.

With timely correction and prevention of metabolic crises, adherence to and strict control of pathogenetic therapy and symptomatic treatment, the prognosis for life is favorable.

With timely initiated treatment, symptoms are generally absent. The lack of therapy in total BD and MCD leads to neurological complications, including mental retardation, impaired consciousness up to coma, and pharmacoresistant epilepsy.

One of the most important approaches to the early diagnosis of BD and MCD is newborn screening. BD and MCD meet the criteria for inclusion in newborn screening and are performed in many countries worldwide, including the Russian Federation.

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

1. Anisimova Inga Vadimovna, PhD in Medical Sciences, Head of the Department of Medical Care Organization, Research Centre for Medical Genetics, medical geneticist, member of the Association of Medical Geneticists.

2. Baydakova Galina Viktorovna, PhD in Biological Sciences, Leading Researcher at the Laboratory of Inherited Metabolic Diseases, Research Centre for Medical Genetics, member of the Russian Society of Medical Geneticists.

3. Baranov Alexander Alexandrovich, Academician of the RAS, Professor, Doctor of Medical Sciences, Honorary President of the Union of Pediatricians of Russia, Advisor to the Head of the Research Institute of Pediatrics and Children's Health of the Central Clinical Hospital of the RAS, Professor at the Department of Pediatrics and Pediatric Rheumatology, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University), Chief freelance pediatric specialist of the Ministry of Health of Russia.

4. Baranova Polina Vladimirovna, Researcher at the Core Facility "Metabolom", Research Centre for Medical Genetics.

5. Bushueva Tatyana Vladimirovna, Doctor of Medical Sciences, National Medical Research Center for Children's Health of the Ministry of Health of the Russian Federation, member of the Children's Health Association and the Association of Medical Geneticists, Professor at the Department of Biochemical Genetics and Inherited Metabolic Diseases, Research Centre for Medical Genetics.

6. Vashakmadze Nato Dzhemberovna, Doctor of Medical Sciences, Head of the Department of Orphan Diseases and Prevention of Disabling Conditions, Research Institute of Pediatrics and Children's Health of the Central Clinical Hospital of the RAS, Professor at the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Ministry of Health of Russia.

7. Voronin Sergey Vladimirovich, PhD in Medical Sciences, Chief Physician of the Research Centre for Medical Genetics, Chief freelance specialist in medical genetics of the Ministry of Health of Russia in the Far Eastern Federal District.

8. Degtyareva Anna Vladimirovna, Doctor of Medical Sciences, Professor, Head of the Department of Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov, Professor at the Department of Neonatology, Institute of Children's Health, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University).

9. Zakharova Ekaterina Yuryevna, Doctor of Medical Sciences, Head of the Laboratory of Inherited Metabolic Diseases, Research Centre for Medical Genetics, member of the Russian Society of Medical Geneticists, member of the Society for the Study of Inborn Errors of Metabolism (SSIEM).

10. Kekeeva Tatyana Nikolaevna, Medical geneticist at the consultative department, Research Centre for Medical Genetics.

11. Krasnoshchekova Nina Alexandrovna, Pediatrician, medical geneticist, Morozov Children's City Clinical Hospital of the Moscow Healthcare Department.

12. Kuzenkova Lyudmila Mikhailovna, Doctor of Medical Sciences, Head of the Department of Psychoneurology and Psychosomatic Pathology, National Medical Research Center for Children's Health of the Ministry of Health of the Russian Federation, member of the Children's Health Association.

13. Kutsev Sergey Ivanovich, Doctor of Medical Sciences, Academician of the RAS, Director of the Research Centre for Medical Genetics, President of the Association of Medical Geneticists.

14. Lobanova Viktoria Sergeevna, Neurologist, epileptologist at the Morozov Children's City Clinical Hospital of the Moscow Healthcare Department, member of the Society for the Study of Inborn Errors of Metabolism (SSIEM).

15. Lobenskaya Anastasia Yuryevna, Head of the Biochemical Laboratory, laboratory geneticist, Diagnostic Center (Medical Genetics) of Saint Petersburg.

16. Mikhailova Svetlana Vitalievna, Doctor of Medical Sciences, Head of the Department, Russian Children's Clinical Hospital of the Pirogov Russian National Research Medical University.

17. Nazarenko Lyudmila Pavlovna, Professor, Doctor of Medical Sciences, Deputy Director for Research and Clinical Work, Head of the Laboratory of Hereditary Pathology, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the RAS, member of the Association of Medical Geneticists.

18. Namazova-Baranova Leila Seymurovna, Academician of the RAS, Professor, D.Sc. (Med.), President of the Union of Pediatricians of Russia; Past President of EPA/UNEPSA; Head of the Research Institute of Pediatrics and Child Health, Central Clinical Hospital of the Russian Academy of Sciences of the Ministry of Science and Higher Education of the Russian Federation, Head of the Department of Faculty Pediatrics, Faculty of Pediatrics, Pirogov Russian National Research Medical University of the Russian Ministry of Health, Chief freelance pediatric specialist in preventive medicine of the Russian Ministry of Health.

19. Nikolaeva Ekaterina Alexandrovna, D.Sc. (Med.), Chief Researcher at the Department of Hereditary and Congenital Diseases with Mental Disorders, Veltischev Research and Clinical Institute of Pediatrics, Pirogov Russian National Research Medical University of the Russian Ministry of Health, Department of Clinical Genetics, Veltischev Research and Clinical Institute of Pediatrics, Pirogov Russian National Research Medical University of the Russian Ministry of Health.

20. Pechatnikova Natalia Leonidovna, Head of the City Center for Orphan and Other Rare Diseases in Children and Adolescents, Morozov Children's City Clinical Hospital of the Moscow Healthcare Department.

21. Selimzyanova Lilia Robertovna, Ph.D. (Med.), Leading Researcher at the Research Institute of Pediatrics and Child Health, Central Clinical Hospital of the Russian Academy of Sciences of the Ministry of Science and Higher Education of the Russian Federation, Associate Professor at the Department of Pediatrics and Pediatric Rheumatology, Sechenov First Moscow State Medical University of the Russian Ministry of Health (Sechenov University).

22. Smirnova Olga Yakovlevna, Medical Geneticist, Senior Researcher at the Department of Standardization and Study of Evidence-Based Medicine Foundations, Research Institute of Pediatrics and Child Health, Central Clinical Hospital of the Russian Academy of Sciences.

23. Strokova Tatyana Viktorovna, D.Sc. (Med.), Professor of the RAS, Head of the Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Clinic of the Federal Research Centre of Nutrition and Biotechnology.

24. Subbotin Dmitry Mikhailovich, Medical Geneticist, Research Center for Medical Genetics named after Academician N.P. Bochkov, Researcher at the Laboratory of Neurogenetics.

25. Sumina Maria Gennadyevna, Head of the Department of Medical Genetic Counseling, Medical Geneticist, Clinical and Diagnostic Center "Maternal and Child Health Protection" of the Sverdlovsk Region.

26. Taran Natalia Nikolaevna, Ph.D. (Med.), Senior Researcher at the Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Federal Research Centre of Nutrition and Biotechnology, Assistant at the Department of Pirogov Russian National Research Medical University of the Russian Ministry of Health.

The authors declare no financial support or conflicts of interest that need to be disclosed.

Appendix A2. Methodology for Developing Clinical Guidelines

These guidelines are intended for use by medical organizations and institutions of federal, territorial, and municipal healthcare management systems, compulsory and voluntary medical insurance systems, and other medical organizations of various legal forms engaged in the provision of medical care.

The clinical guidelines are based on a systematic literature review of 1980–2023 from Medline (PubMed version), Embase (Dialog version) and Cochrane Library databases, utilizing developed protocols of modern international clinical guidelines for the diagnosis, treatment, and management of patients with metabolic diseases.

MPS and MSD belong to rare hereditary disorders, which precludes large cohort and randomized controlled trials; therefore, diagnostic and therapeutic protocols rely solely on expert case studies published over the past two decades.

The evaluation of the quality of evidence and the strength of recommendations for the application of medical technologies was performed in accordance with the standardized scale presented in Tables 1–3.

Target audience for these clinical guidelines:

1. General practitioners (family physicians);

2. Pediatricians;

3. General practitioners;

4. Medical geneticists;

5. Laboratory geneticists;

6. Cardiologists;

7. Pediatric cardiologists;

8. Neurologists;

9. Functional diagnostics physicians;

10. Otorhinolaryngologists;

11. Ophthalmologists;

12. Medical rehabilitation physicians;

13. Otorhinolaryngologist-audiologists;

14. Medical psychologists;

15. Medical students;

16. Residents and postgraduate students.

Table 1. Scale for grading levels of evidence (LE) for diagnostic methods (diagnostic interventions).

Table 2. Scale for grading levels of evidence (LE) for methods of prevention, treatment, medical rehabilitation, including those based on natural therapeutic factors (preventive, therapeutic, and rehabilitative interventions).

Table 3. Scale for grading levels of recommendation (LR) for methods of prevention, diagnosis, treatment, medical rehabilitation, including those based on natural therapeutic factors (preventive, therapeutic, and rehabilitative interventions).

Procedure for updating clinical guidelines

The update mechanism for clinical guidelines provides for their systematic revision at least once every three years, as well as upon the emergence of new evidence-based medicine data regarding the diagnosis, treatment, prevention, and rehabilitation of specific diseases, or in the presence of well-founded additions/comments to previously approved clinical guidelines, but no more frequently than once every 6 months.

Appendix A3. Reference Materials

1. Federal Law No. 323-FZ dated November 21, 2011 (as amended on December 28, 2022) "On the Fundamentals of Healthcare Protection in the Russian Federation" (with amendments and additions, effective from January 11, 2023).

2. Order of the Russian Ministry of Health No. 274n dated April 21, 2022, "On Approval of the Procedure for Providing Medical Care to Patients with Congenital and (or) Hereditary Diseases."

3. Order of the Russian Ministry of Health No. 804n dated October 13, 2017 (as amended on September 24, 2020, with rev. dated October 26, 2022) "On Approval of the Nomenclature of Medical Services".

4. Order of the Russian Ministry of Health No. 203n dated May 10, 2017, "On Approval of Criteria for Assessing the Quality of Medical Care."

5. Order of the Russian Ministry of Health No. 1183n dated December 20, 2012 (as amended on September 4, 2020) "On Approval of the Nomenclature of Positions of Medical and Pharmaceutical Workers".

6. Order of the Russian Ministry of Health No. 700n dated October 7, 2015 (as amended on December 9, 2019) "On the Nomenclature of Specialties of Specialists with Higher Medical and Pharmaceutical Education".

7. Order of the Russian Ministry of Health No. 103n dated February 28, 2019 (as amended on June 23, 2020) "On Approval of the Procedure and Timeframes for the Development of Clinical Practice Guidelines, Their Revision, the Standard Template of Clinical Practice Guidelines, and Requirements for Their Structure, Composition, and Scientific Validity of Information Included in Clinical Practice Guidelines".

8. Information on medicinal products: https://grls.rosminzdrav.ru/

9. International Statistical Classification of Diseases and Related Health Problems (ICD-10).

Collection of biomaterial for diagnostics in blood spots

Preparation for blood collection

Preparation for blood collection is standard: medical personnel must follow sterility rules by wiping the puncture site with a sterile wipe, using disposable lancets and gloves.

Obtaining a dried blood spot

As a rule, for obtaining dried blood spots [72], blood is collected from a finger; venous blood collected from an EDTA-containing tube may also be used (depending on the test kits used).

In newborns, blood for neonatal screening and retesting is collected exclusively from the heel, and in exceptional cases from a finger (if heel blood collection is impossible).

The puncture site (heel, finger) should be treated with a sterile wipe, then blotted with a dry sterile wipe. The first drop formed at the puncture site is not used—it is wiped away with a dry cotton swab. Subsequent drops are applied one by one into the circles on the absorbent membrane of the test card. The circles on the card must be saturated evenly and all the way through, without white spots on the back of the card (Fig. 1D3). Obtaining a single spot requires 80–100 µl of blood. After applying the blood to the test card, the sample is allowed to dry completely for at least 2 hours at room temperature, without touching other test cards. After drying, the test cards are placed into individual packaging, ensuring the blood spots do not touch or overlap one another.

Figure 1G3. Dried blood spots on a test form.

For molecular genetic and biochemical studies outside of neonatal screening, in addition to droplet application, the required volume of whole blood may be applied to the test form using a semi-automatic dispenser, for example, from a tube containing the analyzed biological material.

Transportation of test forms is carried out at a temperature not exceeding 25°C [MR SPK].

Algorithm of actions for medical personnel during blood sample collection:

• wash hands (hygienic level), put on gloves;

• wash the patient's hands (the infant's heel, if blood is collected from the heel);

• wipe the puncture area with a sterile wipe, blot with a dry sterile wipe; - puncture with a sterile disposable lancet;

• remove the first drop of blood with a sterile dry swab;

• gently press to obtain a second drop of blood;

• apply the test card perpendicularly to the drop of blood and saturate it completely through with blood;

• similarly apply 6–8 drops onto the test card; the appearance of the blood spots must be identical on both sides;

• dry the test card in a horizontal position on a clean, grease-free surface for at least 4 hours without heat treatment and direct sunlight;

• package the test cards in a clean envelope so that the blood spots do not touch.

Specifics during infusion therapy

Certain patients may receive infusion therapy or blood component transfusions, which can affect test results. For example, plasma transfusions may yield false-negative results because the enzymes being determined are present in both plasma and blood cells. It is recommended to perform blood collection for enzyme and DNA diagnostics no earlier than 6–7 days after plasma transfusion and 7–10 days after blood component transfusion.

Blood collection is not permitted:

• immediately after the patient undergoes infusion therapy;

• immediately after an exchange blood transfusion.

Storage and transportation of biological material

Dried blood spot samples can be stored in a regular refrigerator compartment at +4°C until shipment. The storage period prior to shipment must not exceed 7 days. The temperature during transportation of the biomaterial should be from 0°C to +10°C. Stored longer or at a higher temperature, enzyme activity may decrease even within the normal range, leading to false-positive results.

Appendix B. Physician Action Algorithms

If BT or HCSD is detected, it is necessary to call the family in for a consultation and examine the infant to decide on hospitalization. Newborns with suspected BT and HCSD (elevated C5OH concentration) are subject to hospitalization in a neonatal pathology or intensive care unit (depending on their condition), where blood is taken for a retest, determination of biotinidase and holocarboxylase synthetase enzyme activities, as well as urine collection for organic acid analysis (samples are sent to the reference center). Depending on the enzyme activities and the results of the urine organic acid test, the diagnosis of BT and HCSD can be confirmed at the biochemical level or another diagnosis can be established (Fig. 1B). After receiving positive results, the newborn must be immediately referred to a specialist (medical geneticist, pediatrician) to prescribe pathogenetic therapy, perform confirmatory diagnostics, as well as provide education and counseling to the family and arrange for follow-up care. Confirmatory diagnostics include molecular genetic testing of the BTD and HLCS genes, which is performed at the reference center.

Appendix C. Patient Information

What are BT and HCSD?

Multiple carboxylase deficiency (MCD) is an inherited metabolic disorder caused by impaired biotin metabolism in the body. Biotin is a B-complex vitamin that enters the human body through diet. Biotin is essential for the production (synthesis) and function of other enzymes—carboxylases: propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and acetyl-CoA carboxylase. These carboxylases play a vital role in many biological processes involving proteins, fats, and carbohydrates.

In the absence or impaired function of biotinidase enzymes (biotinidase deficiency, BD) or holocarboxylase synthetase (holocarboxylase synthetase deficiency, HCSD), biotin cannot be assimilated by the body. This leads to biotin deficiency and, consequently, impaired production of all biotin-dependent carboxylases, i.e., multiple carboxylase deficiency (MCD). Furthermore, toxic substances (organic acids) accumulate in the body, damaging the central nervous system (CNS) and other organs.

Why does the disease occur?

BD and HCSD are hereditary diseases associated with disruptions (mutations) in the BTD and HLCS genes, respectively.

Genes represent a sequence of DNA. They contain instructions for "making" proteins or RNA (ribonucleic acid). Genes are located on chromosomes. Humans have 23 pairs of chromosomes. One chromosome with its corresponding set of genes is inherited from the mother, and the second from the father.

The HLCS gene is located on chromosome 21, and the BTD gene is located on chromosome 3.

When a mutation occurs in a gene, it can no longer serve as an "instruction" for creating a protein (in this case, the enzymes biotinidase and holocarboxylase synthetase). Thus, the resulting enzymes function improperly or are not produced in the body at all.

Both diseases, BD and HCSD, are inherited in an autosomal recessive manner, meaning that both parents are carriers of the mutation but are not sick because they have a second copy of the healthy gene. A child with BD or HCSD inherits one copy of the "diseased" gene from the father and one from the mother. The disease manifests only if two copies of the damaged gene are present. The risk of having a child with BD or HCSD in this case is 25% (Fig. 1B). Boys and girls are affected with equal frequency. A disease carrier inherits only one "damaged" copy: either from the father or from the mother. Carriers are not sick and have no signs of the disease.

How does BD manifest?

Profound BD begins to manifest between 3 and 5.5 months of age. Most often, the first symptoms of BD are seizures and muscle weakness. The main symptoms also include: gait and coordination disorders, psychomotor retardation, breathing rhythm disorders, partial (patchy) hair loss or alopecia, conjunctivitis, skin manifestations (rash, dermatitis, seborrhea), as well as recurrent viral or fungal infections. The CNS is particularly vulnerable in BD because biotinidase enzyme activity in the human brain is very low. This can lead to a number of neurological disorders, such as hearing and vision loss.

The severity of clinical manifestations depends on the level of biotinidase activity in the blood. Partial BD has a milder form of the disease and manifests under stress conditions (e.g., prolonged fasting, infection, physical exertion).

How does HCSD manifest?

The symptoms of HCSD are very similar to those of BD. The disease usually manifests in the first few hours of life, days, or weeks after birth. The initial symptoms are refusal to eat, repeated vomiting, increased drowsiness or irritability, muscle weakness, and dermatitis. Without treatment, the disease progresses rapidly, leading to epileptic seizures, cerebral edema, and psychomotor and physical developmental delays in the child.

How are BD and HCSD diagnosed?

Based on clinical symptoms (seizures, hair loss, muscle weakness, skin manifestations), physicians may suspect a diagnosis of BD and HCSD. The diagnosis is confirmed when biotinidase and holocarboxylase synthetase activity decreases in blood plasma or serum. In urine, the concentrations of β-hydroxyisovalerate, β-methylcrotonylglycine, β-hydroxypropionate, methylcitrate, 3-hydroxyisovalerylcarnitine (C5OH), lactate (lactic acid), and tiglylglycine are elevated. To confirm the diagnosis of BD and HCSD, DNA analysis is performed by testing the HLCS and BTD genes for mutations.

All families with BD and HCSD must undergo medical genetic counseling and receive comprehensive information from a geneticist regarding the risk of recurrence of the disease in the family, as well as discuss all issues related to the inheritance of the disease with a specialist. In Russia, medical genetic consultation clinics operate in every region.

For the purpose of early (prenatal) detection of hereditary pathology in families with a diagnosed case of BD and HCSD, prenatal and preimplantation diagnostics are performed. The couple must first undergo screening for carrier status of mutations in the HLCS and BTD genes responsible for these diseases by contacting specialized diagnostic laboratories and medical centers. Prenatal diagnostics are performed using molecular genetic methods by examining DNA extracted from chorionic villus biopsy samples and/or amniotic fluid cells and/or fetal blood. Preimplantation genetic diagnosis is performed prior to the in vitro fertilization (IVF) procedure before the embryo is implanted (introduced into the mother's uterus). Nevertheless, prenatal diagnosis of treatable conditions such as BD and HCSD is possible, but ethically questionable. The decision to perform it must be made after a detailed discussion of all risks with the family.

How are patients with BD and HCSD treated?

With timely initiation of treatment, the prognosis for patients with BD and HCSD is favorable. The mainstay of treatment for BD and HCSD is lifelong administration of high doses of biotin. In most cases of early treatment initiation, all symptoms resolve, preventing the development of severe neurological complications.

Appendix G. Assessment Scales, Questionnaires, and Other Evaluation Tools

Appendix G1. Assessment of infant motor development using the Alberta Infant Motor Scale

Russian title: Alberta Infant Motor Scale.

Original title: Alberta Infant Motor Scale (AIMS).

Source (official developer website, validation publication): Piper MC, Pinnell LE, Darrah J, Maguire T, Byrne PJ. Construction and validation of the Alberta Infant Motor Scale (AIMS). Can J Public Health. 1992;83:S46-50.

Type (underline):

- rating scale.

Purpose: The Alberta Infant Motor Scale (AIMS), an observational rating scale, was designed to measure gross motor maturation in infants from birth through independent walking.

Content (template): Based on literature, 58 skills were generated and categorized into four positions: prone, supine, sitting, and standing. Each skill is assigned a specific score.

Key (interpretation): Scores are summed to provide an overall assessment of motor development and deviation from/concordance with normal development in percentiles.

Alberta Infant Motor Scale

Total score: ______ percentile: ______

Notes/Recommendations:

INFANT MOTOR DEVELOPMENT RATING SCALE

Percentile values