3-Hydroxy-3-methylglutaryl-CoA lyase deficiency: clinical guidelines

List of Abbreviations

3-HMGCLD — 3-hydroxy-3-methylglutaryl-CoA lyase deficiency;

3-HMGCL — 3-hydroxy-3-methylglutaryl-CoA lyase;

C5OH — 3-hydroxyisovalerylcarnitine;

CoA — coenzyme A;

CT — computed tomography;

MRI — magnetic resonance imaging;

MS/MS — tandem mass spectrometry.

Terms and Definitions

Metabolic crisis — a critical, life-threatening condition triggered by adverse factors that cause enhanced cellular catabolism with the accumulation of toxic derivatives, manifesting as acute encephalopathy, vomiting episodes, 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

3-Hydroxy-3-methylglutaryl-CoA lyase deficiency (3-HMGCLD) is a progressive inherited metabolic disorder based on a defect in the HMGCL gene encoding the enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (3-HMGCL) and associated with impaired ketone body and leucine metabolism [1] (OMIM 246450). The disease is characterized by episodes of metabolic ketoacidosis with the accumulation of leucine metabolites detected by quantitative and qualitative analysis of urine organic acids (3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-hydroxyisovaleric, 3-methylglutaconic, adipic, suberic, and sebacic acids), as well as elevated blood acylcarnitine concentrations (3-hydroxyisovalerylcarnitine).

Synonym: 3-hydroxy-3-methylglutaric aciduria.

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

3-HMGCL deficiency (3-HMGCLD) is an autosomal recessive disorder characterized by impaired leucine catabolism and ketone body utilization. Characteristic laboratory findings include tissue accumulation and increased urinary excretion of leucine catabolism intermediates, such as 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-hydroxyisovaleric, and 3-methylglutaconic acids. Urine also shows increased concentrations of dicarboxylic acids, such as adipic, suberic, and sebacic acids, particularly during metabolic decompensation (crisis) [2, 3].

The disease is caused by impaired functioning of the enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase), which is located in the mitochondrial matrix and peroxisomes in the liver, brain, muscles, leukocytes, and fibroblasts. It is known that HMG-CoA lyase functions in two metabolic pathways (Fig. 1). In the liver, HMG-CoA lyase, together with another enzyme—3-hydroxy-3-methylglutaryl-CoA synthase—participates in the formation of ketone bodies from fatty acids [4]. Ketone bodies, such as acetoacetate and beta-hydroxybutyrate, are important sources of energy for extrahepatic organs, particularly the brain, during periods of insufficient energy supply. In other tissues, HMG-CoA lyase catalyzes the final reaction in leucine catabolism: the cleavage of 3-hydroxy-3-methylglutaryl-CoA into acetoacetate and acetyl-CoA [1, 4]. Thus, according to its pathogenesis, HMG-CoA lyase deficiency can be classified into two groups of inborn errors of metabolism: inherited disorders of ketogenesis and organic acidurias.

Figure 1. Biochemical pathways involving the enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase) in leucine and ketone body metabolism.

The disease results from homozygous and compound heterozygous mutations in the HMGCL gene, mapped to the long arm of chromosome 1 (1q36.11) [5]. According to ClinVar data, to date there are at least 79 pathogenic and likely pathogenic variants of the HMGCL gene leading to impaired HMG-CoA lyase function: nonsense variants (20/79; 25.3%), frameshift variants (20/79; 25.3%), splice-site variants (12/79; 15.2%), and missense variants (6/79; 7.6%). All described missense substitutions are known to cause a loss of enzyme activity by more than 95% [6].

A high incidence of HMG-CoA lyase deficiency is observed in Saudi Arabia, where the most prevalent variant is c.122G>A (p.Arg41Gln) [7]. HMG-CoA lyase deficiency is also among the most common organic acidurias detected in the populations of Brazil and Portugal [8, 9]. Other common worldwide variants include the nonsense substitution c.109G>T (p.Glu37Ter, p.E37X), first described in Portugal, and the deletion c.504_505delCT (p.S169LfsX8) [10, 11]. According to literature data, variants c.122G>A and c.109G>A are responsible for 87% and 94% of HMG-CoA lyase deficiency cases, respectively, in countries such as Saudi Arabia, Portugal, and Spain [12]. The number of individuals with confirmed variants in the HMGCL gene is estimated at approximately 200 worldwide [13].

Currently, genotype-phenotype correlation in HMG-CoA lyase deficiency has not been established [14].

The pathophysiology of the disease has been only partially studied. Patients with HMG-CoA lyase deficiency suffer, on the one hand, from a shortage of ketone bodies as an alternative energy source and, on the other hand, from the accumulation of toxic metabolites of leucine catabolism: 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-hydroxyisovaleric, and 3-methylglutaconic acids. All these compounds provoke attacks of metabolic ketoacidosis and exert toxic effects on tissues. The excretion of these metabolites occurs in the urine as carnitine conjugates, causing secondary carnitine deficiency [15], which can lead to hypoglycemia, hepatomegaly, cardiomyopathy, and hyperammonemia [16]. Administration of L-carnitine to patients with HMG-CoA lyase deficiency can prevent secondary carnitine deficiency [17] and reduce the level of oxidative DNA and lipid damage caused by the accumulation of 3-hydroxy-3-methylglutaric and 3-methylglutaric acids [18]. Fasting, infections, and immunization stimulate catabolic processes, accompanied by an increased concentration of free amino acids, including leucine, and exacerbating the entire chain of molecular pathogenic processes.

1.3 Epidemiology of the Disease or Condition

The prevalence of HMG-CoA lyase deficiency varies depending on ethnicity and geographical location. Thus, HMG-CoA lyase deficiency is among the most common organic acidurias detected in the populations of Saudi Arabia, Brazil, and Portugal [7–9]. According to a systematic review of 211 cases of HMG-CoA lyase deficiency in 2017, mortality due to the disease is 16% [19]. The incidence of HMG-CoA lyase deficiency averages 1:1,000,000 live births [20]. In the Russian Federation, the frequency of the disease has not been determined.

1.4 Features of Coding the Disease or Condition

According to ICD-10, HMG-CoA lyase deficiency belongs to Class IV: Endocrine, nutritional and metabolic diseases.

ICD-10: E71.1 — Other disorders of branched-chain amino-acid metabolism.

ICD-11: 5C52.02 — Ketone body metabolism disorders.

OMIM: 246450

ORPHA code: 20

1.5 Classification of the disease or condition

Classification is absent.

1.6 Clinical presentation of the disease or condition

According to a 2020 systematic study, in 42.4% of cases the first symptoms manifest already in the neonatal period [13]. In 80% of cases, HMG-CoA lyase deficiency manifests within the first year of life [13], but cases of adult-onset disease are known [21–23]. The latest manifestation was registered in a patient at 54 years of age [23].

The clinical phenotype is heterogeneous. In the majority of patients, the disease presents with episodes of metabolic acidosis (triggered by fasting periods, infections, or excessive physical exertion), non-ketotic/hypoketotic hypoglycemia, cyanosis, vomiting, seizures, hypotonia, and lethargy [20, 24]. In its manifestations, the disease resembles Reye syndrome, as it proceeds as liver dysfunction with hypoglycemia [5, 17, 25–27]. Patients with HMG-CoA lyase deficiency also exhibit hepatomegaly, hyperammonemia, fatty liver disease, and elevated serum transaminases (aspartate aminotransferase, alanine aminotransferase) [5]. The elevation of liver enzymes is often mild, but several patients have been reported with concentrations rising up to 1400 U/L [5, 19, 28]. Cases of acute and recurrent pancreatitis are known [28, 29].

Patients with HMG-CoA lyase deficiency exhibit a number of progressive neurological complications, including hypotonia, speech delay, developmental delay, learning disabilities, epileptic seizures, spasticity, and encephalopathy [17, 19]. Magnetic resonance imaging (MRI) reveals nonspecific periventricular and deep white matter changes, cerebral atrophy, demyelination, cerebral ischemia [17, 30], as well as chronic subdural hematomas, subdural hygromas, and bilateral occipital porencephaly [13]. Cases of corticospinal tract involvement [31] and bilateral basal ganglia lesions [32] are known. One patient was described with macrocephaly with widespread white matter changes manifesting as hypodensity on computed tomography (CT), a "doll-like face", a prominent forehead, and a depressed nasal bridge [33].

Cardiovascular involvement includes cases of dilated cardiomyopathy with arrhythmia in three patients with HMG-CoA lyase deficiency, with fatal outcomes in two cases [34, 35]. It has been suggested that cardiomyopathy is the result of impaired ketogenesis, intracellular fatty acid accumulation, and secondary carnitine deficiency [35]. Left ventricular involvement was diagnosed in one patient [35].

The main clinical and biochemical signs of HMG-CoA lyase deficiency according to the literature are presented in Table 1.

Table 1. Main clinical and laboratory findings in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG-CoA lyase deficiency).

↑ = increase in concentration / elevated.

2. Diagnostics, medical indications and contraindications for diagnostic methods

Please note that, in accordance with the requirements for developing clinical guidelines, each recommendation statement must be accompanied by its recommendation grade and evidence level based on the grading systems for the levels of evidence certainty (LEC) and recommendation credibility levels (RCL). For many statements, the RCL and LEC will be low due to the lack of high-design clinical studies addressing them. Despite this, they are essential elements of patient evaluation for establishing a diagnosis and selecting treatment strategies.

Criteria for establishing diagnosis and conditions.

The diagnosis of HMG-CoA lyase deficiency is established based on a combination of anamnestic data, clinical findings, and laboratory test results (biochemical and molecular genetic analysis). Detection of elevated concentrations of 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-methylglutaconic, 3-hydroxyisovaleric, adipic, suberic, and sebacic acids upon quantitative and qualitative urinary organic acid analysis; along with elevated blood acylcarnitine concentrations—specifically 3-hydroxyisovalerylcarnitine (C5OH)—allow for a clinical diagnosis.

Identification of biallelic pathogenic variants in the HMGCL gene via DNA diagnostics confirms the diagnosis at the molecular genetic level.

Differential diagnosis is carried out with all conditions accompanied by Reye-like syndrome, including mitochondrial $\beta$-oxidation defects, systemic carnitine deficiency, glutaric aciduria type II, other organic acidurias (isovaleric aciduria, 3-hydroxy-3-methylglutaric aciduria, and others), as well as consequences of perinatal central nervous system injury, spastic-hyperkinetic form of cerebral palsy, congenital malformations of the brain, and consequences of intrauterine infection.

2.1 Complaints and Anamnesis

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

• positive family history (similar symptoms in siblings of the proband, consanguineous marriage);

• disease manifestation before 1 year of age;

• feeding difficulties, refusal to eat;

• lethargy, weakness, fatigue;

• diarrhea;

• depressed level of consciousness ranging from drowsiness to coma;

• seizures;

• muscular hypotonia ("floppy infant syndrome");y

• cardiomyopathy and cardiac arrhythmias;

• behavioral changes.

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 3-HMGCoA lyase deficiency:

• hepatomegaly;

• psychomotor retardation;

• muscular hypotonia;

• spastic hemiparesis or tetraplegia;

• macro- or microcephaly;

• gait disturbance (cerebellar ataxia);

• cyanosis.

2.3 Laboratory Diagnostics

The main laboratory methods to confirm the diagnosis of 3-HMGCoA lyase deficiency include quantitative and qualitative analysis of urinary organic acids, blood acylcarnitine profiling, and molecular genetic testing of the HMGCL gene. These studies are performed in specialized genetic laboratories.

Comments: C5OH concentrations may be within the normal range. The algorithm for the differential diagnosis of 3-HMGCoA lyase deficiency with other conditions associated with elevated C5OH levels is presented in Appendix B.

Comments: characteristic of 3-HMGCoA lyase deficiency is an increased concentration of 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-methylglutaconic, 3-hydroxyisovaleric, adipic, suberic, and sebacic acids in urine.

Comments: this test is necessary to verify the diagnosis at the molecular genetic level. According to ClinVar data, to date, at least 79 pathogenic and likely pathogenic variants of the HMGCL gene leading to impaired 3-HMGCoA lyase enzyme function are known: nonsense variants (20/79; 25.3%), frameshift variants (20/79; 25.3%), splice site variants (12/79; 15.2%), and missense variants (6/79; 7.6%). Common pathogenic variants include c.122G>A, (p.Arg41Gln), c.109G>T (p.Glu37Ter, p.E37X) and c.504_505delCT (p.S169LfsX8) [7, 10, 11].

Currently, genotype-phenotype correlation in 3-HMGCoA lyase deficiency has not been established [14].

Comments: patients may exhibit anemia, thrombocytosis, leukocytosis, leukopenia, the timely detection of which allows for the prevention of complications and is obviously an integral part of the initial examination and subsequent follow-up. If relevant changes are detected, the issue of referring the patient for an initial consultation with a hematologist should be considered.

Comments: laboratory precursors of a metabolic crisis include a tendency toward metabolic acidosis with base deficit, and increased renal excretion of organic acids.

Comments: patients with 3-HMGCoA lyase deficiency present with non-ketotic/hypoketotic hypoglycemia, hyperammonemia, and elevated blood lactate levels [19].

Comments: elevation of liver transaminases (aspartate aminotransferase, alanine aminotransferase) is often mild, but more significant changes have been reported in a few patients with 3-HMGCoA lyase deficiency [5, 19, 28].

2.4 Instrumental Diagnostics

Comments: brain MRI reveals nonspecific changes in the periventricular and deep white matter, cerebral atrophy, demyelination, brain ischemia [17, 30], as well as chronic subdural hematomas, subdural hygromas, and bilateral occipital porencephaly [13]. Cases of corticospinal tract involvement [31] and bilateral basal ganglia lesions [32] are known. Macrocephaly with widespread white matter changes manifesting as hypodensity on CT, "doll-like face", prominent forehead, and depressed nasal bridge was described in one patient [33]. General anesthesia is performed according to indications.

Comments: cases of dilated cardiomyopathy with arrhythmia have been reported in three patients with 3-HMGCoA lyase deficiency, resulting in death in two cases [34-35]. It has been suggested that cardiomyopathy is the result of impaired ketogenesis, accumulation of intracellular fatty acids, and secondary carnitine deficiency [36]. Left ventricular involvement was diagnosed in one patient [35].

If changes are detected on instrumental studies or if cardiovascular involvement is clinically suspected, patients require an initial consultation with a cardiologist/pediatric cardiologist.

If necessary, when indications for the study are present, Holter ECG monitoring and/or ambulatory blood pressure monitoring is performed (according to the relevant clinical guidelines for cardiac arrhythmias and/or arterial hypertension).

Comments: patients may present with hepatomegaly, which is one of the characteristic signs of a group of inherited metabolic disorders, a sign of acute decompensation, and a marker of disease severity. Cases of acute and recurrent pancreatitis have been reported in patients with 3-HMGCoA lyase deficiency [28, 29].

2.5 Other Procedures

2.5.1 Specialist Consultations and Monitoring

Monitoring patients with an established diagnosis of 3-MCCD requires a multidisciplinary approach, as these disorders are characterized by multi-organ and multisystem involvement, which in turn necessitates coordinated management by specialists from various fields. Initial and follow-up consultations are indicated by a geneticist, neurologist, cardiologist, ophthalmologist, otolaryngologist-audiologist, hematologist, otorhinolaryngologist, gastroenterologist, psychiatrist, pediatrician / general practitioner / family physician, as well as other specialists for patients with 3-MCCD who have functional impairments of the corresponding organs and systems.

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

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

Comments: patients with 3-MCCD present with a number of progressive neurological complications, including hypotonia, speech delay, developmental delay, learning disabilities, epileptic seizures, spasticity, and encephalopathy [17, 19]. The Alberta Infant Motor Scale is provided in Appendix G1.

Comments: patients with 3-MCCD exhibit speech delay, hyperactivity, and learning difficulties [32]. Patients need testing using approved methods to determine 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 3-MCCD require differential diagnosis with Reye-like syndrome, including mitochondrial fatty acid β-oxidation disorders, systemic carnitine deficiency, glutaric aciduria type 2, other organic acidurias (isovaleric aciduria, 3-hydroxy-3-methylglutaric aciduria, etc.), as well as with the consequences of perinatal nervous system lesions, the spastic-hyperkinetic form of cerebral palsy, congenital malformations of the brain, and the consequences of intrauterine infection. In cases of ketosis combined with hypoglycemia, conditions accompanied by ketotic hypoglycemia should be considered—endocrine disorders (e.g., adrenal insufficiency) and inherited disorders of carbohydrate metabolism (e.g., glycogen synthase deficiency, glycogen storage diseases). When ketoacidosis is combined with hyperammonemia, differential diagnosis with organic acidemias is performed.

The algorithm for the differential diagnosis of 3-MCCD with other disorders associated with altered C5OH levels is shown in Fig. 1B of Appendix B.

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

Due to the small number of described cases, there are no uniform treatment protocols for 3-MCCD. Based on the pathogenesis of the disease, the main therapeutic approaches include the administration of L-carnitine (levocarnitine)-containing medications, as well as a diet restricting leucine and/or natural protein with the addition of specialized leucine-free nutritional products. Some specialists also recommend restricting dietary fat intake.

The main principles of therapy in 3-MCCD are aimed at preventing metabolic decompensation (metabolic crisis), reducing the formation of toxic organic acids, preventing toxic damage to the brain tissues and internal organs while ensuring normal growth and development in children. Antiepileptic drugs and other types of symptomatic therapy are prescribed according to indications.

3.1 Pathogenetic treatment

Patients with 3-MCCD and low free carnitine levels are prescribed medications containing L-carnitine (levocarnitine) at a dosage of 75-100 mg/kg/day for pathogenetic treatment [30]. The excretion of toxic 3-hydroxy-3-methylglutaric, 3-methylglutaric, and 3-hydroxyisovaleric acids occurs in the urine as carnitine conjugates, which causes secondary carnitine deficiency [15] that can lead to hypoglycemia, hepatomegaly, cardiomyopathy, and hyperammonemia [16]. Administration of L-carnitine to patients with 3-MCCD can prevent secondary carnitine deficiency [17] and reduce the level of oxidative DNA and lipid damage caused by the accumulation of 3-hydroxy-3-methylglutaric and 3-methylglutaric acids [18].

Comments: the diet is prescribed by a dietician, neurologist, geneticist, pediatrician, or gastroenterologist. Protein intake (1.5-2 g/kg/day) and fat intake (6.3 g/kg/day) must be restricted [30, 47]; a ketogenic diet is contraindicated. Studies show that patients with 3-MCCD on a low-protein diet normalize clinical and biochemical liver parameters [5]. Patients with 3-MCCD must also avoid prolonged fasting, and in case of fever and/or vomiting, carbohydrate intake must be increased. When urinary ketone bodies are elevated, dextrose** infusions are administered.

3.1.1 Management of patients during a metabolic crisis

If a metabolic crisis threatens or develops, treatment must be initiated immediately. Therapeutic measures are aimed at stopping the production and accumulation of toxic organic compounds and excreting them from the body. Treatment tactics for children during a crisis include correction of metabolic acidosis and water-electrolyte disturbances, as well as energetic support. In the event of a threatened or developing metabolic crisis, emergency hospitalization is necessary for immediate intensive care and prevention of life-threatening complications.

Treatment of a metabolic crisis is carried out in the intensive care unit by an anesthesiologist-resuscitologist, as well as other medical specialists (neurologist, nephrologist, gastroenterologist, etc.). During crises, intravenous administration of dextrose** is necessary even with normal glucose levels to maintain its concentration at the upper limit of the norm, which helps suppress ketogenesis. If the pH drops (<7.1), sodium bicarbonate** is administered as a bolus according to the instructions for use regarding the relief of decompensated metabolic acidosis, followed by continuous infusion. 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 of patients diagnosed with 3-MCCD is carried out primarily by a neurologist, cardiologist, or gastroenterologist. Treatment of disease complications is performed in accordance with clinical guidelines for the respective pathology.

Patients with 3-MCCD present with a number of progressive neurological complications, including hypotonia, speech delay, developmental delay, learning disabilities, epileptic seizures, spasticity, and encephalopathy [17, 19]. The Alberta Infant Motor Scale is provided in Appendix G1.

Cases of dilated cardiomyopathy with arrhythmia have been reported in relation to the cardiovascular system [34, 35]. A case of left ventricular involvement in a patient with 3-HMGCoA lyase deficiency has been documented [35]. It has been hypothesized that cardiomyopathy results from impaired ketogenesis, accumulation of intracellular fatty acids, and secondary carnitine deficiency [35]. All patients require initial and follow-up examinations (evaluations, consultations) by a cardiologist and a pediatric cardiologist at least once every 6 months.

3.3 Surgical Treatment

Not applicable.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

Specific rehabilitation is not required for patients with 3-HMGCoA lyase deficiency. The rehabilitation plan for patients with 3-HMGCoA lyase deficiency may include sessions with a psychologist, stays in specialized sanatoriums, as well as social adaptation involving specialists and social workers, and courses of medical massage.

5. Prevention and Dispensary Observation

5.1 Prevention

Patients with 3-HMGCoA lyase deficiency should receive routine vaccinations in accordance with the national immunization schedule, including vaccination against influenza and rotavirus infection, observing the age restrictions for immunobiological drugs (J07: Vaccines) against the background of strict adherence to therapeutic prescriptions to build an effective immune response and prevent metabolic crises. Vaccination should preferably be carried out under the supervision of specialists experienced in managing patients with inborn errors of metabolism. In the post-vaccination period, vaccinated individuals should be closely monitored with repeated body temperature measurements. If a fever >38.0°C is detected, antipyretic therapy (paracetamol** or ibuprofen**) must be prescribed immediately.

Comments: families with children diagnosed with 3-HMGCoA lyase deficiency require medical genetic counseling to determine genetic risk. As with other autosomal recessive disorders, the recurrence risk of 3-HMGCoA lyase deficiency for each pregnancy is 25%. Prenatal diagnosis of treatable conditions is possible but ethically controversial; the decision to perform it must be made after a detailed discussion of all risks with the family.

5.2 Dispensary monitoring

Patients with a confirmed diagnosis of 3-HMGCoA lyase deficiency must undergo a comprehensive annual health check-up. Patient monitoring requires a multidisciplinary approach because this condition is characterized by multi-organ and multisystem involvement, which in turn necessitates coordinated management by specialists from various fields. To ensure comprehensive therapy and timely adjustment thereof when necessary, initial and follow-up evaluations (examinations, consultations) are indicated by a geneticist, neurologist, otorhinolaryngologist, ophthalmologist, pediatrician/general practitioner/family physician, rehabilitation specialist, as well as other specialists for patients with 3-HMGCoA lyase deficiency presenting with functional impairments of the respective 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: 3-HMGCoA lyase deficiency is characterized by elevated urinary concentrations of 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-methylglutaconic, 3-hydroxyisovaleric, adipic, suberic, and sebacic acids. The frequency of testing depends on the patient's clinical condition.

Comments: Blood C5OH levels resulting from treatment should be close to age-specific reference values.

Comments: the complete blood count with differential should be monitored at least once a year.

Comments: the general clinical blood chemistry panel is monitored at least once a year.

Comments: laboratory precursors of a metabolic crisis include a tendency toward metabolic acidosis with base deficit, and increased renal excretion of organic acids.

Comments: the consultation is performed at least once every 6 months. Patients with 3-HMGCoA lyase deficiency exhibit a range of progressive neurological complications, including hypotonia, speech delay, developmental delay, learning disabilities, epileptic seizures, spasticity, and encephalopathy [17, 19]. The Alberta Infant Motor Scale is provided in Appendix G1.

6. Organization of Medical Care

Indications for scheduled hospitalization in a medical organization:

1. Provision of diagnostics 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 basic clinical and laboratory parameters of the pathological process, absence of the need for prolonged medical observation (day 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

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.

The prognosis of the patient's condition and mental development level depends on the severity of the disease, the presence of stroke-like episodes, the involvement of internal organs in the pathological process (kidneys, heart, pancreas), as well as the timing of the initiation of specialized therapy and the quality of treatment capable of preventing episodes of metabolic decompensation.

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

1. Inga V. Anisimova, Candidate of Medical Sciences, Head of the Department of Medical Care Organization, Research Centre for Medical Genetics; geneticist

2. Galina V. Baydakova, Candidate of Biological Sciences, Head of the Metabolom Shared Research Facility, Leading Research Fellow at the Laboratory of Hereditary Metabolic Diseases, Academician N.P. Bochkov Research Centre for Medical Genetics, member of the Russian Society of Medical Genetics

3. Alexander A. Baranov, Academician of the Russian Academy of Sciences, 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 Child Health, Petrovsky National Research Centre of Surgery, Professor at the Department of Pediatrics and Pediatric Rheumatology, Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University), Chief Freelancer Pediatrician of the Ministry of Health of Russia

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

5. Tatyana V. Bushuyeva, Doctor of Medical Sciences, National Medical Research Center for Children's Health of the Ministry of Health of the Russian Federation, member of the Union of Pediatricians of Russia.

6. Nato D. Vashakmadze, Doctor of Medical Sciences, Head of the Department of Orphan Diseases and Prevention of Disabling Conditions, Research Institute of Pediatrics and Child Health, Petrovsky National Research Centre of Surgery, Professor at the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Ministry of Health of Russia

7. Anna V. Degtyareva, Doctor of Medical Sciences, Professor, Head of the Department of Pediatrics, Institute of Neonatology and Pediatrics, Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Professor at the Department of Neonatology, Institute of Child Health, Sechenov First Moscow State Medical University, member of the Council of the Russian Society of Neonatologists

8. Ekaterina Yu. Zakharova, 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)

9. Victoria S. Kakaulina, Neurologist, Epileptologist Morozovskaya Children's City Clinical Hospital, member of the Society for the Study of Inborn Errors of Metabolism (SSIEM)

10. Tatiana N. Kekeeva, Head of the Medical Genetics Department, Morozovskaya Children's City Clinical Hospital, geneticist

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

12. Lyudmila M. Kuzenkova, 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 Union of Pediatricians of Russia

13. Marina V. Kurkina, Researcher at the Laboratory of Inherited Metabolic Diseases, Research Centre for Medical Genetics

14. Sergey I. Kutsev, Academician of the RAS, Doctor of Medical Sciences, Director of the Research Centre for Medical Genetics, President of the Association of Medical Geneticists of Russia

15. Anastasia Yu. Lobenskaya — Head of the Biochemistry Laboratory, Laboratory Geneticist, Diagnostic Center (Medical Genetics), Saint Petersburg

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

17. Lyudmila P. Nazarenko, 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. (Medicine), President of the Union of Pediatrists of Russia; Past President of EPA/UNEPSA; Head of the Research Institute of Pediatrics and Child Health at the Petrovsky National Research Centre of Surgery; Head of the Department of Faculty Pediatrics, Pediatric Faculty, 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. Ekaterina A. Nikolaeva, Doctor of Medical Sciences, Head of the Department of Clinical Genetics, Veltischev Research and Clinical Institute of Pediatrics, Pirogov Russian National Research Medical University, member of the Russian Society of Medical Genetics, member of the Association of Medical Geneticists

20. Natalia L. Pechatnikova, 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. Liliya R. Selimzyanova, Candidate of Medical Sciences, Head of Department, Research Institute of Pediatrics and Child Health, Petrovsky National Research Centre of Surgery, Associate Professor at the Department of Pediatrics and Pediatric Rheumatology, Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University), Associate Professor at the Department of Faculty Pediatrics, Pediatric Faculty, Pirogov Russian National Research Medical University of the Ministry of Health of Russia, member of the Union of Pediatricians of Russia

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

23. Strokova Tatyana Viktorovna, D.Sc. (Medicine), 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 at the Bochkov Research Centre for Medical Genetics, Member of the Association of Medical Geneticists

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. Natalia N. Taran, Candidate of Medical Sciences, Senior Research Fellow at the Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Federal Research Center of Nutrition and Biotechnology, Assistant at the Department, Pirogov Russian National Research Medical University of the Ministry of Health of Russia.

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 review of the 1976–2022 literature from Medline (PubMed version), Embase (Dialog version), and Cochrane Library databases, using established protocols of current international clinical guidelines for the diagnosis, treatment, and management of patients with metabolic diseases.

3-HMG-CoA lyase deficiency is a rare hereditary disease, which excludes the possibility of conducting large cohort and randomized controlled trials; therefore, only expert case studies published over the past two decades are used to establish diagnostic and therapeutic protocols.

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. Ophthalmologists;

8. Otorhinolaryngologists;

9. Hematologists;

10. Pediatric cardiologists;

11. Neurologists;

12. Functional diagnostics physicians;

13. Medical psychologists;

14. Medical students;

15. Residents and postgraduate students.

Table 1. Level of Evidence (LoE) grading scale for diagnostic methods (diagnostic interventions)

Table 2. Scale for assessing levels of reliable evidence (LRE) for prevention, treatment, and rehabilitation methods (preventive, therapeutic, rehabilitation interventions)

Table 3. Grade of Recommendation (GoR) scale for prevention, diagnostic, treatment, and rehabilitation methods (preventive, diagnostic, 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 Protecting the Health of Citizens in the Russian Federation" (with rev. and add., effective January 11, 2023).

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

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

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

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

Sampling of biological material for dried blood spot testing

Preparation for blood collection.

Preparation for blood collection is standard: medical personnel must observe sterility rules by wiping the puncture site with an antiseptic solution and using disposable lancets and gloves.

Obtaining a dried blood spot

As a rule, to obtain dried blood spots [50], capillary blood from a finger is collected; venous blood collected from an EDTA vacuum tube may also be used (depending on the assay 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 cleaned with a sterile wipe soaked in 70% ethanol**, and then blotted dry with a sterile gauze pad. The first drop formed at the puncture site is discarded — it is wiped away with a dry cotton swab. Subsequent drops are successively applied to the circles on the absorbent test form. The circles on the card must be saturated evenly and completely, with no white spots on the reverse side of the card (Fig. 1G2). A volume of 80–100 µl of blood is required to obtain a single spot. After applying 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 in individual packaging, ensuring that blood spots do not touch or overlap with one another.

Figure 1G2. Dried blood spots on a test card.

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 child's heel if blood is collected from the heel);

• wipe the puncture area with a sterile wipe moistened with 70% ethanol**, and 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;

• apply 6–8 drops to the test form in a similar manner; the appearance of the blood spots should 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 measured are present in both plasma and blood cells. Blood collection for enzyme and DNA testing must be performed no earlier than 6–7 days after a blood plasma transfusion and 7–10 days after a blood component transfusion.

Blood collection is not permitted:

• immediately after the patient undergoes infusion therapy;

• immediately after an exchange blood transfusion procedure.

Storage and transportation of biological material

Dried blood spot samples can be stored in a standard refrigerator compartment at +4°C until shipment. The storage period prior to shipment must not exceed 7 days. The temperature during transportation of the biological material must be between 0°C and +10°C. If stored longer and at a higher temperature, enzyme activity may decrease even in normal ranges, leading to false-positive results.

Appendix B. Physician Action Algorithms

If 3-HMG-CoA lyase deficiency is detected, the family must be called in for a consultation and the child examined to address the issue of hospitalization. Since elevated C5OH levels are not specific to this disorder, additional tests and confirmatory diagnostics are required (Fig. 1B). The prognosis is favorable and largely depends on the timing of diagnosis and initiation of treatment.

Neonates with suspected 3-HMG-CoA lyase deficiency are subject to admission to a neonatal pathology unit or intensive care unit (depending on their clinical status), where blood is collected for retesting, and urine samples are collected for urinary organic acid analysis (samples are sent to a reference center). Depending on the urine organic acid results, the diagnosis of 3-HMG-CoA lyase deficiency may be biochemically confirmed or an alternative diagnosis established. Upon receiving positive results, the neonate must be immediately referred to a specialist (medical geneticist, pediatrician) to prescribe etiopathogenetic therapy, perform confirmatory diagnostics, provide family education and counseling, and arrange for ongoing follow-up. Confirmatory diagnostics are performed at a reference center, and the diagnosis is confirmed at the molecular genetic level by identifying biallelic pathogenic variants in the HMGCL gene.

Figure 1B. Neonatal screening algorithm for the detection of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (3-HMG-CoA lyase deficiency) and other disorders associated with elevated levels of 3-hydroxyisovaleroylcarnitine (C5OH): holocarboxylase synthetase deficiency and 3-methylcrotonyl-CoA carboxylase deficiency.

Appendix C. Patient Information

Why does the disease occur?

3-Hydroxy-3-methylglutaryl-CoA lyase deficiency (3-HMG-CoA lyase deficiency) is a rare hereditary disease caused by disruptions (mutations) in the HMGCL gene.

Genes represent a DNA sequence that encodes 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 HMGCL gene is located on chromosome 1. When a mutation occurs in the HMGCL gene, it can no longer serve as an "instruction" for producing the 3-HMG-CoA lyase enzyme. Consequently, the resulting enzyme functions abnormally or is not produced in the body at all.

3-HMG-CoA lyase deficiency is inherited in an autosomal recessive manner. This means that a child develops the disease only if both parents are carriers of the condition and, while remaining healthy themselves, transmit two copies of the "damaged" gene (the mutated gene) to the child. A carrier inherits only one "damaged" copy: either from the father or the mother. Carriers do not have the disease and show no signs of it. The risk of having a child with 3-HMG-CoA lyase deficiency in a family where both parents are carriers of the "mutated" gene is 25% for each pregnancy (Fig. 1C). Boys and girls are affected with equal frequency.

All families affected by the disease must undergo medical genetic counseling and receive comprehensive information from a medical geneticist regarding the recurrence risk of the condition in the family, and discuss all inheritance-related issues with a specialist. Medical genetic counseling services are available in every region of Russia.

Figure 1C. Inheritance pattern of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (3-HMG-CoA lyase deficiency). With two copies of the "damaged" gene ("r") inherited from both carrier parents, the risk of having a child with 3-HMG-CoA lyase deficiency is 25% ("rr").

What metabolic processes are impaired in 3-HMG-CoA lyase deficiency?

In 3-HMG-CoA lyase deficiency, the 3-hydroxy-3-methylglutaryl-CoA lyase (3-HMG-CoA lyase) enzyme is non-functional. Normally, this enzyme is involved in the breakdown of dietary leucine. Leucine is one of the 20 amino acids that make up proteins. Patients with 3-HMG-CoA lyase deficiency lack this enzyme, and leucine is not fully degraded. When such patients consume protein-rich food, high levels of leucine accumulate in the blood and are converted into hazardous toxic substances that harm the body. Additionally, the 3-HMG-CoA lyase enzyme has a secondary function: it is used during fat breakdown to produce ketone bodies. When the body lacks sufficient glucose, ketone bodies serve as the primary "fuel" for our organs. Patients with 3-HMG-CoA lyase deficiency have low levels of ketone bodies and, consequently, lack a backup energy source. As a result, harmful compounds can accumulate, causing the blood to become excessively acidic (ketoacidosis), which impairs tissue function, particularly within the central nervous system. Ketoacidosis is a life-threatening pathological condition requiring immediate medical intervention.

How does 3-HMG-CoA lyase deficiency manifest?

There are no symptoms at birth. In 80% of cases, the disease manifests within the first year of life, although cases with symptom onset in adulthood are also known. The symptoms of 3-HMG-CoA lyase deficiency are highly diverse. Affected infants often become irritable, refuse to eat, and exhibit diarrhea, nausea, vomiting, muscle weakness, lethargy, and fatigue. Symptoms frequently appear after prolonged fasting, infection, or physical exertion. Patients with 3-HMG-CoA lyase deficiency may develop neurological complications, including developmental delay, speech delay, and epilepsy. Patients with 3-HMG-CoA lyase deficiency may also present with cardiomyopathy accompanied by arrhythmias, as well as liver involvement (hepatomegaly, fatty liver disease).

How is 3-HMG-CoA lyase deficiency diagnosed?

Initial diagnosis of the disease is carried out by a pediatrician. The diagnosis is established based on the clinical presentation, urinalysis (elevated levels of 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-hydroxyisovaleric, 3-methylglutaconic, adipic, suberic, and sebacic acids), and blood test (elevated concentration of 3-hydroxyisovalerylcarnitine). The diagnosis is confirmed at the molecular-genetic level upon detection of pathogenic variants in the HMGCL gene.

How are patients with 3-HMGCLD treated?

The main principle of therapy for 3-HMGCLD is the prescription of medications containing levocarnitine if patients exhibit low levels of free carnitine. Antiepileptic drugs are prescribed according to indications. Patients are advised to avoid prolonged fasting and a high-fat (ketogenic) diet, as well as to restrict protein intake (1.5–2 g/kg/day) and fat intake (6.3 g/kg/day).

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

- score

- questionnaire

- other

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

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

Key (interpretation): The scores are summed, providing an overall assessment of motor development and deviation/conformity to normal development in percentiles.

Alberta Infant Motor Scale

Total score: ______ percentile: ______

Notes/Recommendations:

INFANT MOTOR DEVELOPMENT RATING SCALE

Percentile values