In this article, we have studied the frequencies of carriers for rare metabolic recessive diseases in 320 Brazilian patients and found 205 occurrences of 172 different variants harbored by 138 different genes in 156 patients. It represents 0.64 occurrence per patient in this cohort. Based on our data and the Hardy–Weinberg equation, we estimate an overall frequency of recessive metabolic diseases to be 10.96/10,000 people, or approximately 0.11%.
The majority of the variants are harbored by genes associated with multisystemic involvement, which widens clinical and social burden of these conditions. Several of these conditions may manifest with acute crises that often require critical care and may involve seizures (64%), acidosis (32%), hypoglycemia (20%) or encephalopathy (14%). The majority of the diseases may also present with long-term complications, such as growth abnormality (66%), intellectual disability (55%) and other complications addressed in Table 2.
It is noteworthy that almost one fifth (18%) of these genes may present with facial dysmorphisms, which is a clinical characteristic generally regarded as non-suggestive of metabolic diseases by clinicians. We also found unexpectedly high frequency of muscle (80%) and ophthalmologic involvement (70%) in the genes harboring P/LP variants. These latter involvements may require specific clinical measurements and should not be neglected in the follow-up of these patients.
We have used the HPO database to estimate the clinical impact of the genes found in our study and discussed above. This approach, though, presents some limitations: HPO tends to be highly inclusive even for rare manifestations of diseases; this fact may have overestimated the multisystemic impact of the diseases. On the other hand, some diseases may be neglected; for instance, CUBN gene was not associated with any HPO term studied, though it is known that the conditions associated with this gene may present with growth anomaly, intellectual disability, renal involvement, etc (OMIM#261100).
We have estimated the frequency of rare recessive metabolic diseases to be 10.96 per 10,000 people. Other studies using different methodologies have estimated variable frequencies of metabolic disorders: 4.0 (40/100,000) , 12,76 (1/784)  or 15.6 (66/42,257)  per 10,000 people. These estimates, though, are usually challenging especially if they are based on biochemical-based newborn screening programs because several factors may influence the results, including the scope of diseases screened, deaths occurring before a diagnosis is made, ease of access for screening programs, diagnosis confirmation methods, specific regional clinical interests . Additionally, the frequency of these diseases may vary widely in different population groups due to founder effects, endogamy, nonrandom mating, and cultural, religious, social and/or geographical isolation . For example, in endogamous societies this rate can be as high as 8.4% in highly consanguineous populations .
Considering the relevant clinical impact of several metabolic diseases, the relatively common combined frequencies and the possibility to improve clinical outcome with early diagnoses, several countries have established newborn screening programs aiming rapid diagnosis and treatment for clinically-actionable metabolic diseases. We have used the Newborn Screening ACT Sheets and Algorithms, from ACMG, to map the metabolic diseases potentially identified by newborn screening programs and we estimate the frequency of these diseases to be 2.93 per 10,000 people. Figure 1 helps to understand the magnitude of the estimated frequency of diseases potentially identified by newborn screening in the universe of predicted frequency of all metabolic disorders. If our estimates are correct, we believe that 26.6% (2.92/10.96) of patients with metabolic disorders may potentially be detected by neonatal screening. Amino acid studies would have the potential to identify 33.6% (0.98/2.92) of potentially detectable diseases, while acylcarnitine analysis the potential to identify 31.8% (fatty acid oxidation disorders: 0.49/2.92 + organic acidemias: 0.44/2.92) and a small fraction (0.7%; 0.02/2.92) would be detected by assays for galactosemia. An important amount of detectable diseases rely on specific tests for lysosomal storage disorders (34.2%; 1.0/2.92) that are generally not widely available in neonatal screening programs.
Our study presents several limitations. The first refers to variant classification and pathogenicity presumption: several variants were classified as likely pathogenic based solely on ACMG criteria but were not definitively proven to be pathogenic (disease-causing); while this fact that may have overestimated the carrier frequencies by falsely classifying some variants as P/LP, we took precautions to standardize variant classification by strictly using ACMG classification criteria uniformly for all sequence variants. On the other hand, we may have excluded several variants that are not yet recognized to cause disease, variants not detected by NGS and missense hypomorphic alleles that do not result in a clear and recognizable loss of function.
Another important issue is the limited number of patients, which is an important bias that compromises the direct estimation of single monogenic disease frequencies. To minimize this limitation, we used the strategy of studying groups of diseases (instead of single diseases) to estimate frequencies, then diluting the small sample of patients.
Our study did not consist of asymptomatic patients, which would be an ideal group for carrier screening studies. However, we took precautions to minimize this bias by eliminating P/LP variants associated with autosomal recessive disorders reported in homozygosity or compound heterozygosity (primary findings) from this analysis because our objective was to study carriers for recessive diseases. Therefore, all patients in this study had only monoallelic variants. Nevertheless, we cannot rule out that some of the variants studied in this work may have contributed to the clinical phenotype of the patient, for example, if the disease presents an unknown codominant, digenic or even oligogenic mechanism or if the other allele harbored a pathogenic or likely pathogenic variant not detected by the NGS methodology (such as intronic or regulatory site variants). Another issue regarding heterozygous carriers is that some monoallelic variants may have clinical impacts and health complications. For instance, some genes (including POLG) may be associated with autosomal dominant forms that have a generally milder clinical impact.
Literature regarding carrier frequencies for autosomal recessive metabolic diseases is limited in Latin America. Most of previously published studies are limited to sole or restricted groups of diseases. Additionally, the majority rely on newborn screening findings without molecular confirmation. We hope that our study encourages other studies that aim to better understand the burden of recessive diseases in our developing countries.