Carnitine-acylcarnitine Translocase Deficiency Research Articles

Clinical and genetic analysis of two pedigrees affected with Carnitine-acylcarnitine translocase deficiency due to variant of SLC25A20 gene

To analyze the clinical phenotype and genotypes of two children with Carnitine-acylcarnitine translocase deficiency (CACTD). Two children diagnosed with CACTD at the Gansu Provincial Maternal and Child Health Care Hospital respectively on January 3 and November 19, 2018 were selected as the study subjects. Trio-whole exome sequencing (trio-WES) was carried out, and candidate variants were validated through Sanger sequencing and pathogenicity analysis. Both children were males and had manifested mainly with hypoglycemia. Trio-WES and Sanger sequencing showed that child 1 had harbored compound heterozygous variants of the SLC25A20 gene, namely c.49G>C (p.Gly17Arg) and c.106-2A>G, which were inherited from his father and mother, respectively. Child 2 had harbored homozygous c.199-10T>G variants of the SLC25A20 gene, which were inherited from both of his parents. Among these, the c.106-2A>G and c.49G>C variants were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.49G>C (p.Gly17Arg), c.106-2A>G, and c.199-10T>G variants were classified as likely pathogenic (PM2_supporting+PP3+PM3_strong+PP4), pathogenic (PVS1+PM2_supporting+PM5+PP3), and pathogenic (PVS1+PM2_supporting+PP3+PP5), respectively. Combined with their clinical phenotype and genetic analysis, both children were diagnosed with CACTD. Above finding has provided a basis for their treatment as well as genetic counseling and prenatal diagnosis for their families.

The significance of acylcarnitine ratio indices in diagnosing carnitine-acylcarnitine translocase deficiency.

The significance of acylcarnitine ratio indices in diagnosing carnitine-acylcarnitine translocase deficiency.

Acylcarnitine ratio indices in diagnosing carnitine-acylcarnitine translocase deficiency in newborns.

Acylcarnitine ratio indices in diagnosing carnitine-acylcarnitine translocase deficiency in newborns.

Increased acylcarnitine ratio indices in newborn screening for carnitine-acylcarnitine translocase deficiency shows increased sensitivity and reduced false-positivity.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive metabolic disorder of mitochondrial long-chain fatty acid oxidation. Newborn screening via tandem mass spectrometry (MS/MS) technology enables early diagnosis. However, previous analyses of MS/MS data of patients showed that some results were misdiagnosed because they did not show typical acylcarnitine profiles of CACT deficiency. This study aimed to identify additional indices to assist the diagnosis of CACT deficiency. To evaluate the acylcarnitine profile and the acylcarnitine ratios of individuals with CACT deficiency, the MS/MS data of 15 patients diagnosed via genetic testing were retrospectively analysed. The sensitivity and false-positive rates of primary acylcarnitine markers and ratio indices were validated using the data from 28,261 newborns and 53 false-positive cases. Additionally, the MS/MS data of 20 newborns carrying the c.199-10T>G mutation in SLC25A20 and 40 normal controls were compared to verify whether the carriers had abnormal acylcarnitine concentrations. The acylcarnitine profiles from 15 patients were classified into three categories using C12, C14, C16, C18, C16:1, C18:1, and C18:2 as the primary diagnostic markers. The first category represented a typical profile (P1-P6). The second category for patients P7 and P8 showed a significant decrease in the C0 level and a normal concentration of long-chain acylcarnitines. The third category for patients P9-P15 showed the presence of interfering acylcarnitines. The second and third categories may have been misdiagnosed. An acylcarnitine ratio analysis showed that C14/C3, C16/C2, C16/C3, C18/C3, C16:1/C3, and C16:1-OH/C3 were significantly increased in all 15 patients. The verification of 28,261 newborn screening results showed that the false-positive rate of ratios, except for (C16 + C18)/C0, was lower than that of acylcarnitine indices (0.02-0.08% vs. 0.16-0.88%). None of the single long-chain acylcarnitines could separate patients from the false-positive cases; however, all ratios produced good discrimination between the two groups. Based on the primary acylcarnitine markers alone, CACT deficiency can be misdiagnosed in newborn screening. The ratios of the primary markers (C16 + C18:1)/C2, C16/C2, C16:1/C3, and C16:1-OH/C3 can facilitate the diagnosis of CACT deficiency, thereby increasing sensitivity and reducing false-positivity.

In Silico Analysis of the Structural Dynamics and Substrate Recognition Determinants of the Human Mitochondrial Carnitine/Acylcarnitine SLC25A20 Transporter.

The Carnitine-Acylcarnitine Carrier is a member of the mitochondrial Solute Carrier Family 25 (SLC25), known as SLC25A20, involved in the electroneutral exchange of acylcarnitine and carnitine across the inner mitochondrial membrane. It acts as a master regulator of fatty acids β-oxidation and is known to be involved in neonatal pathologies and cancer. The transport mechanism, also known as "alternating access", involves a conformational transition in which the binding site is accessible from one side of the membrane or the other. In this study, through a combination of state-of-the-art modelling techniques, molecular dynamics, and molecular docking, the structural dynamics of SLC25A20 and the early substrates recognition step have been analyzed. The results obtained demonstrated a significant asymmetry in the conformational changes leading to the transition from the c- to the m-state, confirming previous observations on other homologous transporters. Moreover, analysis of the MD simulations' trajectories of the apo-protein in the two conformational states allowed for a better understanding of the role of SLC25A20 Asp231His and Ala281Val pathogenic mutations, which are at the basis of Carnitine-Acylcarnitine Translocase Deficiency. Finally, molecular docking coupled to molecular dynamics simulations lend support to the multi-step substrates recognition and translocation mechanism already hypothesized for the ADP/ATP carrier.

Successful Management by Selective Embryo in the Carnitine-Acylcarnitine Translocase Deficiency with SLC25A20 C.199-10T>G Variation: The First Case Report from Vietnam and Literature Review

Successful Management by Selective Embryo in the Carnitine-Acylcarnitine Translocase Deficiency with SLC25A20 C.199-10T>G Variation: The First Case Report from Vietnam and Literature Review

One potential hotspot SLC25A20 gene variants in Chinese patients with carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase deficiency (CACT deficiency) is a rare and life-threatening autosomal recessive disorder of mitochondrial fatty acid oxidation caused by variant of SLC25A20 gene. The most prevalent missense variant in the SLC25A20 gene in Asia was c.199-10T > G. Due to the c.199-10T > G variant, CACT deficiency is a severe phenotype. Herein, we present a neonatal case with c.199-10T > G variant in China and analyze the clinical, biochemical, and genetic aspects of 78 patients previously identified with CACT deficiency. The patient presented with a series of severe metabolic crises that rapidly deteriorated and eventually died 3 days after delivery. The sequencing of the patient's genome indicated that he was homozygous for the c.199-10T > G variant. 30 patients were found to have the c.199-10T > G mutation, of which 23 were Chinese and 22 were afflicted by the c.199-10T > G splicing variation. In China, c.199-10T > G allele frequency was 82.6%. In CACT deficiency, prompt recognition and treatment are critical. Our data suggested that c.199-10T > G may be a potential hotspot SLC25A20 gene mutation in the Chinese population. Detection of single nucleotide polymorphism is possible for high-risk patients and parents in China.

Rare Cause of Arrhythmia and Seizures in a Late-Preterm Newborn

Rare Cause of Arrhythmia and Seizures in a Late-Preterm Newborn

Neonatal sudden death caused by a novel heterozygous mutation in SLC25A20 gene: A case report and brief literature review

Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare and life-threatening autosomal recessive disorder of fatty acid β-oxidation (FAO). Most patients with CACTD develop severe metabolic decompensation which deteriorates progressively and rapidly, causing death in infancy or childhood. As CACTD in some patients is asymptomatic or only with some nonspecific symptoms, the diagnosis is easy to be ignored, resulting in sudden death, which often triggers medical disputes. Herein, we report a case of neonatal sudden death with CACTD. The neonate showed a series of severe metabolic crisis, deteriorated rapidly and eventually died 3 days after delivery. Tandem mass spectrometry (MS-MS) screening of dry blood spots before death showed that the level of long-chain acylcarnitines, especially C12-C18 acylcarnitine, was increased significantly, and therefore a diagnosis of inherited metabolic disease (IMD) was suspected. Autopsy and histopathological results demonstrated that there were diffuse vacuoles in the heart and liver of the deceased. Mutation analysis revealed that the patient was a compound heterozygote with c.199–10 T > G and a novel c.1A > T mutation in the SLC25A20 gene. Pathological changes such as heart failure, arrhythmia and cardiac arrest related to mitochondrial FAO disorders are the direct cause of death, while gene mutation is the underlying cause of death.

Screening for neonatal inherited metabolic disorders by tandem mass spectrometry in Guangzhou.

To analyze the screening results for inherited metabolic disorders (IMD) in newborns by tandem mass spectrometry (MS/MS) in Guangzhou.A total of 272 117 newborns in Guangzhou from Jan 2015 to Dec 2020 were screened for IMD by MS/MS in Guangzhou Newborn Screening Center. When the primary screening was positive, the newborns and their mothers were recalled. For those with positive in re-examination, the biochemical and related genetic analysis were required for confirmation. The screening results, clinical characteristics and outcomes of the confirmed cases were retrospectively analyzed and the performance was optimized. Among 272 117 neonates, 1808 (0.66%) cases were positive in primary screening, and 1738 cases (96.13%) were recalled for review. The median clinical diagnosis time was 15 d after birth. A total of 79 cases of IMD were diagnosed, including 23 with aminoacidopathy, 17 with disorder of organic acid metabolism and 39 with fatty acid oxidation disorders, involving 21 diseases. The incidence rate was 1/3444 in newborns, and the positive predictive value of 4.5%. Four false negative cases were found, all of them were citrin deficiency. The common diseases were primary carnitine deficiency (26.6%), methylmalonic aciduria (12.7%) and phenylalanine hydroxylase deficiency (11.4%). The mothers of 32 cases were confirmed, including 30 cases of primary carnitine deficiency, 1 case of isobutyl-coenzyme A dehydrogenase deficiency and 1 case of 3-methylcromaryl coenzyme A carboxylase deficiency. The detection rate was 1/2451 in total population. During the follow-up, most patients remain asymptomatic, except for 5 severe cases who died early (1 case of maple syrup urine disease, 2 cases of isolated methylmalonic acidurmia, and 2 cases of carnitine-acylcarnitine translocase deficiency); and 10 cases of organic acid metabolism disorders developed mild psychomotor developmental retardation. After optimizing the screening indicators, the number of initial screening positives dropped to 903, and the positive predictive value increased to 9.1%, and no confirmed cases were missed. The incidence rate of fatty acid oxidation disorders is high in Guangzhou. A variety of IMD can be effectively screened out by MS/MS, and the screening performance can be improved by optimizing screening indicators.

Identification of c.199-10T>G mutation in SLC25A20 gene related to fatty acid oxidation disorders on a Vietnamese patient

Fatty acid oxidation disorders (FAODS) consist of rare diseases which affect the energy production of the mitochrondria by disrupting the β-oxidation of fatty acid, resulting in energy deficiency and toxic acumulation in the patient’s body. Typical clinical symtoms of FAODS include rhabdomyolysis, myoglobinuria, cardiomyopathy, hypoketotic hypoglycemia and liver dysfunction on the newborns and could lead to mortality in most of the cases. Mutations occur in different genes in the enzymatic pathway of the mitochrondria may cause diffirent types of FAODS.The objective of this study was to screen and identify genetic mutations associated with fatty acid oxidation disorders in Vietnamese patients through whole exome sequencing analysis. The result revealed a reported homozygous c.199-10T>G mutation in the position of 10 nucleotides before the exon 3 of the SLC25A20 gene. The SLC25A20 gene encodes the carnitine acylcarnitine translocase (CACT), which plays an important role in transporting acylcarnitine and carnitine in the mitochondria. Genetic mutations in this gene often lead to carnitine-acylcarnitine translocase deficiency (CACTD) - a rare form of FAODs. By in silico analysis, the c.199-10T>G mutation was predicted as a splite site mutation that could lead to exon skipping during the creation of mature mRNA. Genetic analysis of the patient’s family showed that both parents had the mutation c.199-10T>G in heterozygous form. This result suggests that every mutant allele in the patient is inherited from parents. Our finding not only improved our understanding of the c.199-10T>G mutation in SLC25A20 gene of our patient but also provides important information for future research, diagnosis and genetic counseling of FAOS in Vietnamese patients.

New insights into carnitine-acylcarnitine translocase deficiency from 23 cases: Management challenges and potential therapeutic approaches.

Carnitine acyl-carnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial long-chain fatty-acid transport. Most patients present in the first 2 days of life, with hypoketotic hypoglycaemia, hyperammonaemia, cardiomyopathy or arrhythmia, hepatomegaly and elevated liver enzymes. Multi-centre international retrospective chart review of clinical presentation, biochemistry, treatment modalities including diet, subsequent complications, and mode of death of all patients. Twenty-three patients from nine tertiary metabolic units were identified. Seven attenuated patients of Pakistani heritage, six of these homozygous c.82G>T, had later onset manifestations and long-term survival without chronic hyperammonemia. Of the 16 classical cases, 15 had cardiac involvement at presentation comprising cardiac arrhythmias (9/15), cardiac arrest (7/15), and cardiac hypertrophy (9/15). Where recorded, ammonia levels were elevated in all but one severe case (13/14 measured) and 14/16 had hypoglycaemia. Nine classical patients survived longer-term-most with feeding difficulties and cognitive delay. Hyperammonaemia appears refractory to ammonia scavenger treatment and carglumic acid, but responds well to high glucose delivery during acute metabolic crises. High-energy intake seems necessary to prevent decompensation. Anaplerosis utilising therapeutic d,l-3-hydroxybutyrate, Triheptanoin and increased protein intake, appeared to improve chronic hyperammonemia and metabolic stability where trialled in individual cases. CACTD is a rare disorder of fatty acid oxidation with a preponderance to severe cardiac dysfunction. Long-term survival is possible in classical early-onset cases with long-chain fat restriction, judicious use of glucose infusions, and medium chain triglyceride supplementation. Adjunctive therapies supporting anaplerosis may improve longer-term outcomes.

Carnitine-Acylcarnitine Translocase Deficiency Masked by Extreme Prematurity.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare disorder of long chain fatty acid oxidation with a very high mortality rate due to cardiomyopathy or multiorgan failure. We present the course of a very premature infant with early onset CACT deficiency complicated by multiple episodes of necrotizing enterocolitis, sepsis, and liver insufficiency, followed by eventual demise. The complications of prematurity, potentiated by the overlay of CACT deficiency, contributed to the difficulty of reaching the ultimate diagnosis of CACT deficiency.

Tutorial: Triheptanoin and Nutrition Management for Treatment of Long-Chain Fatty Acid Oxidation Disorders.

Patients with severe long-chain fatty acid oxidation disorders (LC-FAODs) experience serious morbidity and mortality despite traditional dietary management including medium-chain triglyceride (MCT)-supplemented, low-fat diets. Triheptanoin is a triglyceride oil that is broken down to acetyl-coenzyme A (CoA) and propionyl-CoA, which replenishes deficient tricarboxylic acid cycle intermediates. We report the complex medical and nutrition management of triheptanoin therapy initiated emergently for 3 patients with LC-FAOD. Triheptanoin (Ultragenyx Pharmaceutical, Inc, Novato, CA, USA) was administered to 3 patients with LC-FAOD on a compassionate-use basis. Triheptanoin was mixed with non-MCT-containing low-fat formula. Patients were closely followed with regular cardiac and laboratory monitoring. Cardiac ejection fraction normalized after triheptanoin initiation. Patients experienced fewer hospitalizations related to metabolic crises while on triheptanoin. Patient 1 has tolerated oral administration without difficulty since birth. Patients 2 and 3 experienced increased diarrhea. Recurrent breakdown of the silicone gastrostomy tube occurred in patient 3, whereas the polyurethane nasogastric tube for patient 2 remained intact. Patient 3 experiences recurrent episodes of elevated creatine kinase levels and muscle weakness associated with illness. Patient 3 had chronically elevated C10-acylcarnitines while on MCT supplementation, which normalized after initiation of triheptanoin and discontinuation of MCT oil. Triheptanoin can ameliorate acute cardiomyopathy and increase survival in patients with severe LC-FAOD. Substituting triheptanoin for traditional MCT-based treatment improves clinical outcomes. MCT oil might be less effective in carnitine-acylcarnitine translocase deficiency patients compared with other FAODs and needs further investigation.

A novel homozygous missense SLC25A20 mutation in three CACT-deficient patients: clinical and autopsy data

Carnitine-acylcarnitine translocase (CACT) deficiency is a fatty acid ß-oxidation disorder of the carnitine shuttle in mitochondria, with a high mortality rate in childhood. We evaluated three patients, including two siblings, with neonatal-onset CACT deficiency and revealed identical homozygous missense mutations of p.Arg275Gln within the SLC25A20 gene. One patient died from hypoglycemia and arrhythmia at 26 months; his pathological autopsy revealed increased and enlarged mitochondria in the heart but not in the liver.

The use of sodium DL-3-Hydroxybutyrate in severe acute neuro-metabolic compromise in patients with inherited ketone body synthetic disorders

BackgroundKetone bodies form a vital energy source for end organs in a variety of physiological circumstances. At different times, the heart, brain and skeletal muscle in particular can use ketones as a primary substrate. Failure to generate ketones in such circumstances leads to compromised energy delivery, critical end-organ dysfunction and potentially death. There are a range of inborn errors of metabolism (IEM) affecting ketone body production that can present in this way, including disorders of carnitine transport into the mitochondrion, mitochondrial fatty acid oxidation deficiencies (MFAOD) and ketone body synthesis. In situations of acute energy deficit, management of IEM typically entails circumventing the enzyme deficiency with replenishment of energy requirements. Due to profound multi-organ failure it is often difficult to provide optimal enteral therapy in such situations and rescue with sodium DL-3-hydroxybutyrate (S DL-3-OHB) has been attempted in these conditions as documented in this paper.ResultsWe present 3 cases of metabolic decompensation, one with carnitine-acyl-carnitine translocase deficiency (CACTD) another with 3-hydroxyl, 3-methyl, glutaryl CoA lyase deficiency (HMGCLD) and a third with carnitine palmitoyl transferase II deficiency (CPT2D). All of these disorders are frequently associated with death in circumstance where catastrophic acute metabolic deterioration occurs. Intensive therapy with adjunctive S DL-3OHB led to rapid and sustained recovery in all. Alternative therapies are scarce in these situations.ConclusionS DL-3-OHB has been utilised in multiple acyl co A dehydrogenase deficiency (MADD) in cases with acute neurological and cardiac compromise with long-term data awaiting publication. The use of S DL-3-OHB is novel in non-MADD fat oxidation disorders and contribute to the argument for more widespread use.

Cardiac arrest and ventricular tachycardia in a newborn with carnitine–Acylcarnitine translocase deficiency

We report a female infant who was diagnosed to have carnitine–acylcarnitine translocate deficiency (CACTD) and developed ventricular tachycardia followed by cardiac arrest. Following full feeding, the infant developed significant bradycardia, hypothermia, hypotonia, hypoglycemia, metabolic acidosis, and hyperammonemia. The hyperammonemia was successfully treated with continuous renal replacement therapy (CRRT). One day after the CRRT, she developed ventricular tachycardia that was refractory to medical treatment and synchronized cardioversion. She had cardiac arrest that was unresponsive to full cardiopulmonary resuscitation and died. Raising high index of suspicion of CACTD in the deferential diagnosis of infants present with sudden deterioration, hypoglycemia or hyperammonemia is crucial for early recognition and providing appropriate treatment in this highly lethal disorder.

Continuous renal replacement therapy in a preterm neonate with hyperammonemia secondary to a rare disease

We report a newborn baby girl with a rare cause of hyperammonemia, in which a session of continuous renal replacement therapy (CRRT) was performed successfully despite the child's small size. The baby was born at 34 weeks' gestation with a birth weight of 2.2 kg and was referred on day 4 of life with lethargy, bradycardia, hypothermia, hypoglycemia, and metabolic acidosis associated with hyperammonemia. Following the insertion of the GamCath line, CRRT was performed using continuous venovenous hemodialysis modality, Ultraflux AV Paed dialyzer (Fresenius Medical Care), and multiFiltrate pediatric kit. Over 19 h of therapy, serum ammonia level reduced from >700 μmol/L to 103 μmol/L. Next-generation sequencing revealed a homozygous pathogenic variation in SLC25A20 (NM_000387; exon2; c.109C>T; p.R37X), leading to a diagnosis of carnitine-acylcarnitine translocase deficiency. The child succumbed during a subsequent hospital stay. This case instructively informs on the feasibility and efficacy of CRRT in the management of neonatal hyperammonemia.

Diagnostic contribution of metabolic workup for neonatal inherited metabolic disorders in the absence of expanded newborn screening

Inherited metabolic disorders (IMDs) in neonates are a diagnostic and therapeutic challenge for the neonatologist, with the priority being to rapidly flag the treatable diseases. The objective of this study was to evaluate the contribution of targeted metabolic testing for diagnosing suspected IMDs on the basis of suggestive clinical setting or family history in neonates. We conducted an observational study over five years, from January 1st, 2010 to December 31, 2014 in the neonatal intensive care unit (NICU) at Robert Debré University Hospital, Paris, France. We assessed the number of neonates for whom a metabolic testing was performed, the indication for each metabolic test and the diagnostic yield of this selected metabolic workup for diagnosing an IMD. Metabolic testing comprised at least one of the following testings: plasma, urine or cerebrospinal fluid amino acids, urine organic acids, plasma acylcarnitine profile, and urine mucopolysaccharides and oligosaccharides. 11,301 neonates were admitted at the neonatal ICU during the study period. One hundred and ninety six neonates underwent metabolic testing. Eleven cases of IMDs were diagnosed. This diagnostic approach allowed the diagnosis, treatment and survival of 4 neonates (maple syrup urine disease, propionic acidemia, carnitine-acylcarnitine translocase deficiency and type 1 tyrosinemia). In total, metabolic testing was performed for 1.7% of the total number of neonates admitted in the NICU over the study period. These included 23% finally unaffected neonates with transient abnormalities, 5.6% neonates suffering from an identified IMD, 45.4% neonates suffering from a non-metabolic identified disease and 26% neonates with chronic abnormalities but for whom no final causal diagnosis could be made. In conclusion, as expected, such a metabolic targeted workup allowed the diagnosis of classical neonatal onset IMDs in symptomatic newborns. However, this workup remained normal or unspecific for 94.4% of the tested patients. It allowed excluding an IMD in 68.4% of the tested neonates. In spite of the high rate of normal results, such a strategy seems acceptable due to the severity of the symptoms and the need for immediate treatment when available in neonatal IMDs. However, its cost-effectiveness remains low especially in a clinically targeted population in a country where newborn screening is still unavailable for IMDs except for phenylketonuria in 2019.

Epidemiology of rare diseases detected by newborn screening in the Czech Republic.

Presymptomatic detection of patients with rare diseases (RD), defined by a population frequency less than 1 : 2,000, is the task of newborn screening (NBS). In the Czech Republic (CZ), currently eighteen RD are screened: phenylketonuria/hyperphenylalaninemia (PKU/HPA), congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), cystic fibrosis (CF), medium chain acyl-CoA dehydrogenase deficiency (MCADD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), very long chain acyl-CoA dehydrogenase deficiency (VLCADD), carnitine palmitoyl transferase I and II deficiency (CPTID, CPTIID), carnitine-acylcarnitine translocase deficiency (CACTD), maple syrup urine disease (MSUD), glutaric aciduria type I (GA I), isovaleryl-CoA dehydrogenase deficiency (IVA), argininemia (ARG), citrullinemia (CIT), biotinidase deficiency (BTD), cystathionine beta-synthase-deficient homocystinuria (CBSD HCU), and methylenetetrahydrofolate reductase deficiency homocystinuria (MTHFRD HCU). The aim was to analyze the prevalence of RD screened by NBS in CZ. We examined the NBS programme in CZ from 1 January 2010 to 31 December 2017, which covered 888,891 neonates. Dried blood spots were primarily analyzed using fluorescence immuno-assay, tandem mass spectrometry and fluorimetry. The overall prevalence of RD among the neonate cohort was 1 : 1,043. Individually, 1 : 2,877 for CH, 1 : 5,521 for PKU/HPA, 1 : 6,536 for CF (1 : 5,887 including false negative patients), 1 : 12,520 for CAH, 1 : 22,222 for MCADD, 1 : 80,808 for LCHADD, 1 : 177,778 for GA I, 1 : 177,778 for IVA, 1 : 222,223 for VLCADD, 1 : 296,297 for MSUD, 1 : 8,638 for BTD, and 1 : 181,396 for CBSD HCU. The observed prevalence of RD, based on NBS, corresponds to that expected, more precisely it was higher for BTD and lower for MSUD, IVA, CBSD HCU, MCADD and VLCADD. Early detection of rare diseases by means of NBS is an effective secondary prevention tool.