1. Introduction
High-performance genetic testing involving gene panels, whole-exome sequencing (WES), or whole-genome sequencing (WGS) has emerged as a pivotal tool in Mendelian disease diagnostics [1]. Their expanding utility is notably evident in the precise diagnosis of critically ill children admitted to intensive care units [2,3] and has significantly reduced the diagnostic odyssey associated with rare diseases [4,5]; thus, these tests are expected to be routinely incorporated into pediatric medical care [6]. Variable diagnostic yields for suspected inborn errors of metabolism (IEM) and neurogenetic disorders using next-generation sequencing (NGS) technologies range from 16% to 68% [6,7], revealing wide differences among diverse populations and the employed clinical approaches [8]. Moreover, these diagnostic strategies are related to high rates of medical treatment redirection. For example, Wu et al. recommended specific medications or modifications for the clinical management of 45.5% and 81%, respectively, of studied patients after they reached a molecular diagnosis [9]. Other authors have shown that at least one medical management change related to the application of rapid WES was implemented in 52% of critically ill children [10]. In particular, genetic testing is essential in the diagnostic approach of rare diseases, such as IEM, which are monogenic disorders that involve abnormalities in enzymes, transport proteins, or chaperones [11], as well as to delineate or expand the genotypic and phenotypic spectrum underlying these diseases, and even to redirect medical, nutritional, surgical, or palliative management [12,13]. Genetic testing is also considered essential for prescribing some genotype-dependent IEM treatments (e.g., tyrosinemia type I, phenylketonuria, cystic fibrosis, and tetrahydrobiopterin defects) [14,15]. Additionally, WES/WGS can identify the carrier status or the co-occurrence of other monogenic traits [16,17].
Inborn errors of intermediary metabolism (IEiM) are a subgroup of IEM that comprises defects disrupting the metabolic pathways of proteins, carbohydrates, or lipids, leading to the accumulation of toxic substances or deficiency of essential compounds [11]. Before the wide availability of NGS, the cornerstone of the diagnosis of IEiM patients relied on biochemical measurements of their characteristic metabolites in blood and/or urine, which allowed us to establish a diagnosis, i.e., the elevated blood concentration of branched-chain amino acids along with alloisoleucine, and abnormal excretion of urinary alfa-ketoacids are indicative of maple syrup urine disease (MSUD) [18]; however, not all IEiMs can be unequivocally diagnosed by biochemical profiles [19,20,21]. Thus, the advent of NGS has contributed to establishing a definitive diagnosis, especially in patients whose biochemical profile is unspecific, i.e., high blood concentrations of hydroxy-isovalerylcarnitine (C5OH) in patients with seizures, hyperlactatemia, hypoglycemia with a normal acylcarnitine profile, or such cases whose clinical picture is highly suggestive of IEiM but whose biochemical profile is negative [22].
Unfortunately, in low- or middle-income countries, the possibility of performing genetic testing is not available for all patients; for example, we previously reported that only 33.4% of Mexican patients with an IEiM who were admitted to our tertiary referral hospital had access to diagnostic genetic testing, leading to limited data on the genotypic spectrum underlying these rare diseases in our population [23]. Thus, strategies must be designed and implemented in these countries to diminish the diagnostic gap with other high-income countries [24]. Furthermore, to the best of our knowledge, there are still no reports regarding the usefulness of WES analysis in the diagnostic approach of IEiM among Latin American patients.
Herein, we present the results of WES in 95 patients either with biochemically confirmed IEiM or with the clinical and biochemical suspicion of having an uncharacterized IEiM to determine the following: (a) the concordance between the initial biochemical diagnosis and the responsible genotype identified by WES, (b) the genotypic spectrum underlying the IEiM in the studied Mexican patients, (c) the proportion of patients affected by a second monogenic disease (co-occurrence) due to expected, incidental, or secondary findings, and (d) the modifications in medical or nutritional management after WES results in selected cases.
2. Results
2.1. Study Population and WES Diagnostic Yield
Study Group 1 included 69 patients (38 females, and 31 males, mean age ± SD was 10.9 ± 7.4 years) with a well-defined biochemical phenotype indicative of a specific IEiM (Figure 1). Moreover, study Group 2 comprised 26 patients (13 females, 13 males, mean age ± SD was 9.5 ± 6.8 years) with nonspecific alterations suggestive of an IEiM. The overall percentage and number of patients, as well as those classified according to the categorization of IEiM type, are shown in Figure 1. Consanguinity was found in 14.73% (N = 14/95) of the families, and endogamy was documented in 23.15% (N = 22/95) of them.
The overall concordance between the initial biochemical diagnosis and WES results was 72.6% (N = 69/95), with a proportion of 91.3% (N = 63/69) in Group 1, and 23.1% (N = 6/26) in Group 2 (Figure 1). The concordance by study group and by type of disorder are shown in Figure 2A–F. Notably, urea cycle disorders had the highest overall concordance between initial and final diagnosis (94.1%), followed by organic acid (77.4%), amino acid (66.6%), carbohydrate (64.2%), and lipid disorders (33.4%; Figure 2A). In Group 1, patients with urea cycle disorders presented a WES diagnostic yield of 100%. Conversely, the highest WES diagnostic yield in Group 2 was observed in patients with carbohydrate disorders, representing 66%. Overall, the comparisons between the initial biochemical and final WES diagnoses were statistically different (Figure 2D). This was different when comparing between types of disorder in Group 1 since statistical differences were only observed in amino acids, organic acids, carbohydrates, and lipid disorders (Figure 2E). In Group 2, the comparison between types of disorder was statistically different for all of them (Figure 2F).
2.2. The Genotypic Spectrum of IEiM-Positive Cases
In this study, we identified 83 IEiM-relevant variants distributed among 29 different genes, with pathogenic variants being the most commonly found (Table 1).
All of them were already submitted by us to the Leiden Open Variation Database (LOVD) v.3.0 Build 30 (
The highest number of diagnostic genotypes was documented in carbohydrate (9 genotypes in 9 patients, 100%) and lipid disorders (2 genotypes in 2 patients, 100%), followed by organic acid (23 genotypes in 24 patients, 96%), urea cycle (14 genotypes in 16 patients, 87.5%), and amino acid (15 genotypes in 18 patients, 83%) disorders. The WES-positive IEiM landscape reported in the studied population is shown in Figure 3.
2.3. Unsolved Cases
This category included negative (N = 20/95) and inconclusive (N = 6/95) patients, representing 27.4% (N = 26/95) of the included patients, most of whom (N = 20/26) belonged to Group 2 (Figure 1). The primary biochemical biomarkers related to these unsolved cases are shown in Table 3, which highlights that in Group 1, branched-chain amino acids and acylcarnitines were the most common ones (N = 2/6 each, 33.4%). In contrast, in Group 2, acylcarnitines, especially long-chain ones (C16, C18, and C18:1), were the initial biomarkers in 35% of them (N = 7/20 patients).
2.4. Patients with Co-Occurrence of Other Monogenic Diseases
A second monogenic disease was identified in 10.5% (N = 10/95) of our studied population (Table 4). The expected findings were identified in 4/10 patients, as their previous biochemical profile or clinical phenotype strongly suggested the presence of a second monogenic trait. These four patients presented postaxial polydactyly (3bINP-021), craniosynostosis (3bINP-054), cystinosis with unexplainable and progressive renal failure despite adequate cysteamine treatment (3bINP-082), and G6PD deficiency (3bINP-085), which were confirmed when pathogenic variants were identified in the GLI3, FGFR2, COL4A5, and G6PD genes, respectively (Table 4).
Three patients revealed incidental findings in the ABCA4 (3bINP-069), PIKFYVE (3bINP-109), and VCAN genes (3bINP-100), whereas in the three remaining patients, secondary findings were attributed to RET-, TTN-, and MSH6-related disorders (3bINP-045, 047, and 074, respectively).
We documented three novel variants underlying these second autosomal dominant traits (Table 4). The variant NM_000168.6:c.3740_3743dup, or p.(Cys1249AlafsTer3), was found in GLI3, while the variant NM_000179.3:c.2150_2153del, or p.(Val717AlafsTer18), was documented in MSH6, and the variant NM_004385.5:c.3455C>A, or p.(Ser1152Ter), was found in VCAN.
2.5. Syndromic Entities Not Related to IEiM Identified by WES
Two patients in Group 2 presented a nonspecific elevation of acylcarnitine and hyperbeta-alaninemia with a negative result in WES for IEiM but presented additional clinical abnormalities, suggesting syndromic entities responsible for intellectual disabilities not related to IEiM, including heterozygous pathogenic variants in SOX4 and PAFAH1B1 responsible for Coffin-Siris syndrome type 10 (3bINP-001) and lissencephaly type 1 (3bINP-041), respectively.
2.6. Decisions Taken in Medical or Nutritional Management After WES Results
Based on WES results, in 32/95 cases (33.6%), a decision related to treatment had to be made, categorized as follows. (1) Modification of the initial treatment: in 18/32 patients (56.2%), the causes of these changes were discordance between the initial and final diagnosis, or the co-occurrence of a second monogenic trait or syndromic entities unrelated to IEiM, or initial unspecific diagnosis and a negative WES result. (2) Continuation of initial treatment: in 5/32 (15.6%) patients, despite their unsolved (inconclusive or negative) WES result, maintenance of the current treatment was decided due to clinical improvement. (3) No specific treatment was provided before or after WES because the initial unspecific biochemical findings were not confirmed in 9/32 (28.1%) patients (Table 5).
3. Discussion
This study represents the first cohort of Mexican patients to assess the efficacy of WES in diagnosing IEiM. Overall, the WES diagnostic yield for both study groups was 72.6%, demonstrating that WES analysis facilitated the identification of the genotype responsible for IEiM diagnosis across our entire patient cohort. This study revealed a higher diagnostic yield than previously reported studies assessing IEM, i.e., 38.7% in Canadian patients (N = 12/31) [25] or 59% (N = 83/141) of Spanish newborns with a positive IEM screening result [26].
In Group 1, due to the previously well-defined biochemical phenotype, the concordance between the initial biochemical diagnosis and the final molecular diagnosis was remarkably high, reaching 91.30% (N = 63/69 patients), which contrasted with the substantially lower concordance rate (23.1%) documented in patients in Group 2 who presented unspecific metabolic alterations in amino acid and acylcarnitine profiles or other laboratory parameters (Figure 1). This demonstrates the fact that when a patient presents a specific biochemical profile, the probability of a positive WES result could be high. However, patients with unspecific biochemical profiles are even more in need of a WES test to discover or discard the presence of an IEiM or other genetic entity. A better diagnostic performance of NGS-based strategies supported on a previous detailed phenotypic delineation performed before genotyping in other monogenic traits has also been demonstrated for the heterogeneous group of mitochondrial diseases, i.e., a higher WES concordance was achieved in patients harboring a suggestive score for these disorders (49%, N = 29/59 patients) than in those lacking this previous clinical evaluation (28.8%, N = 17/59) [27]. This finding supports the importance of considering each patient’s previous complete clinical and biochemical assessment before performing WES analysis to improve the overall IEiM diagnostic yield. Additionally, as suggested by other groups [10,28], to increase the WES diagnostic yield in patients affected by a suspected monogenic disorder, all our included patients were first evaluated by clinical geneticists, in addition to the participation of molecular biologists, biochemists, bioinformaticians, nutritionists, and physicians highly trained in the diagnosis and management of inherited metabolic diseases.
The higher concordance of WES reached in study Group 1 supports the notion that biochemical tests are still highly accurate tools for diagnosing IEiM [12], especially for urea cycle disorders, organic acidurias, and amino acid disorders, which showed a WES diagnostic concordance of 100%, 95.6%, and 90%, respectively (Figure 2B,E). Moreover, no discrepancies were observed between the initially assigned biochemical phenotype and the responsible genotype in the 63 patients in Group 1 (Figure 1 and Table 2). Therefore, our results suggest that biochemical tests should be performed immediately in patients with a suspected diagnosis of IEiM for prompt initiation of specific treatments to limit organ damage, especially brain sequelae [29]. Although NGS-based technologies can be time-consuming and expensive and their interpretation remains challenging in some cases [10], they have proven to be a reliable second-tier newborn screening (NBS) methodology by reducing false-positive results, facilitating the timely resolution of the case, and, in some cases, suggesting a more appropriate or specific diagnosis than that initially obtained by mass spectrometry [12].
We found that 6.3% (N = 6/95) of patients had inconclusive WES results. This percentage is lower than that reported by other authors in genetically heterogeneous diseases, such as neuromuscular disorders (21.9%, N = 9/41 [30]) and developmental epileptic encephalopathy (21.9%, N = 31/141 [31]), which could be related to the previously specific biochemical delineation available for most of our patients (N = 69/95; Figure 1).
Moreover, five patients in Group 1 presented inconclusive WES results, attributed to monoallelic pathogenic genotypes for HLCS- and ACADM-related disorders (N = 2/6, patients 3bINP-036 and 052) and biallelic genotypes for VUS DBT- and GALK1-related disorders (N = 3/6, patients 3bINP-076, 089, and 094), which could explain the previously biochemically diagnosed autosomal recessive IEiM (Table 3). Monoallelic HLCS genotypes seem to be infrequent findings in holocarboxylase synthetase deficiency (OMIM #253270), as reported recently in a small sample of Chinese patients biochemically confirmed with this IEiM, where Sanger sequencing revealed biallelic HLCS pathogenic genotypes in all the participants [32]. However, at least one affected patient with an apparent monoallelic HCLS genotype has been described along with a paracentric inversion of chromosome 21 disrupting the second HLCS allele [33]. Additionally, to the best of our knowledge, proven pathogenic deep intronic variants have not been described in the HCLS gene [34], although several gross deletions and duplications encompassing more than one exon have been described (ClinVar:
Regarding the other monoallelic case with suspicion of medium-chain acyl-CoA dehydrogenase deficiency (MCADD, 3bINP-052), it is known that in European populations, such as Portuguese (77.9% of Gypsy origin) [36] and German [37] ones, Sanger sequencing identified biallelic diagnostic ACADM genotypes in 100% of analyzed patients; instead, the identification of monoallelic ACADM genotypes by traditional sequencing approaches seems to be common in Asian populations, as it has been identified in 8.7% (N = 2/23) of Chinese patients biochemically confirmed with MCADD [38] and in 14.3% of MCADD Japanese patients detected by NBS, even identifying normal ACADM genotypes [39]. Unfortunately, a reliable estimation of monoallelic ACADM genotypes in Latino-derived MCADD populations is lacking. As our patient, 3bINP-052, bearing the p.(Lys329Glu) ACADM allele (rs77931234), which has been identified in 80% of European-origin MCADD patients [40], had a typical biochemical MCADD acylcarnitine profile consisting of elevated levels of hexanoylcarnitine (C6), octanoylcarnitine (C8), and decanoylcarnitine (C12), further identification of a second pathogenic allele via other molecular approaches seems plausible. This patient was initially identified by an abnormal NBS result that revealed elevated blood concentrations of C6, C8, and C12, and was referred to our metabolic center to confirm those results. We found the same metabolic pattern suggestive of MCADD; thus, immediate treatment recommendations were started, consisting of frequent meals and avoidance of formulas with medium-chain triglycerides, along with strict medical follow-up in our clinic. At the time of this study, the patient was five years old and had no symptomatology or metabolic crisis associated with MCADD.
Searching for an eventual second pathogenic allele in these two previously described patients could be addressed in the future by applying long-read whole-genome sequencing or RNA-seq methodologies [41], as demonstrated across various monogenic conditions, including autosomal recessive metabolic disorders [42,43]. In particular, RNA-seq has been demonstrated to increase the diagnostic yield of these disorders by 10%–16% compared with WES alone [41].
Additionally, our patients with inconclusive or even negative WES results could be candidates for performing a later reanalysis of their WES data, as it has been estimated that this reassessment 1–3 years after the initial report may increase the diagnostic yield by 3–15% [41]. Additionally, for those genetic conditions in which a copy number variant (CNV) has been implicated, chromosomal microarray analysis can allow the identification of the other variant [41]. Unfortunately, this approach was unsuccessful in identifying the expected second pathogenic HLCS allele in our patient, 3bINP-036 (Table 3).
With respect to the three patients in Group 1 bearing inconclusive biallelic VUS genotypes, additional future strategies, such as in vitro or in vivo functional studies, may be warranted to reclassify these missense VUSs [41,44], considering the highly suggestive metabolic findings observed in these patients, or simply by awaiting the description of other affected patients bearing these same alleles. Moreover, due to the evident biochemical profile (elevated blood concentrations of branched-chain amino acids, including alloisoleucine) and the clinical phenotype highly suggestive of MSUD in patients 3bINP-076 and 094 (Table 3), along with the clinical improvement by specific medical and nutritional treatments observed in both cases and by considering the severity and potentially lethal nature of this disease, the medical decision was to maintain these treatments. The same criterion was applied to patient 3bINP-089 bearing two GALK1 missense VUSs, which seems explain the high blood concentrations of galactose; thus, medical and nutritional treatments were sustained.
Remarkably, the only inconclusive case in Group 2 (patient ID 3bINP-012; Table 3) was possibly related to the very uncommon phenomenon attributed to genome-wide paternal uniparental disomy identified in nearly 0.0002% of patients subjected to clinical exome or chromosomal microarray analyses [45]. This patient is still under study.
With respect to the negative WES results obtained in 20 patients (21.05%; Figure 1 and Table 3), lipid metabolism defects showed the lowest concordance since the genetic cause was demonstrated in only 33.3% (N = 2/6) of the patients. It has been estimated that when WES/WGS are applied as first-tier tests, negative results are common (42.5% to 66%) in most of the studied cohorts of patients [28,44]. It will be essential to consider applying further analysis to determine whether their isolated or persistent nonspecific biochemical abnormalities are due to undetected genetic defects, i.e., promoter or deep intronic variants, CNVs, epistatic–epigenetic mechanisms, low-grade mosaicisms, common pathogenic variants undetected by bioinformatic algorithms, or synergistic oligogenic heterozygosity [8,44], or whether these abnormalities are simply related to environmental factors (exposomes), such as malnutrition, energetic imbalances, prescribed drugs, or infections [1,41,46,47]. In these patients, applying complementary genetic and functional tests, such as trio-WES, WGS, RNA-Seq, epigenomics, metabolomics, proteomics, or optical genome mapping, could be considered in the future. These approaches are crucial for ruling out a genetic etiology for patients with highly suspected inherited disease and negative WES results [12,41,43,44,47]. Remarkably, a single patient in Group 1 had a negative WES result (3bINP-072; Figure 1 and Table 3). This patient was a 29-gestational-week preterm female weighing 1.2 kg at birth who immediately developed respiratory distress syndrome requiring mechanical ventilatory support. She later developed broncho dysplasia, necrotizing enterocolitis requiring ileostomy, retinopathy, and renal tubular acidosis. Biochemically, she presented with hypoglycemia, hyperammonemia, hyperlactatemia, metabolic acidosis, and elevated liver transaminases. An abdominal ultrasound revealed hepatomegaly but no parenchymal structural alterations, splenomegaly, or ascites. A liver biopsy revealed a significant glycogen load in hepatocytes compatible with the diagnosis of glycogen storage disease (GSD) type I, which was not genotypically confirmed by WES. Therefore, other diagnostic possibilities must be ruled out. The patient has been gradually released from the GSD diet.
Concerning the characterized IEiM mutational spectrum described herein, we noted a high proportion of homozygous genotypes (50%), which could be related to the consanguinity (14.73%) and endogamy (23.15%) recorded in our patients. This finding is consistent with our previous report of IEiM families, in which 13.5% consanguinity was documented [23], but is lower than the worldwide rate observed in patients with IEiM (51%) [48]. This finding highlights the importance of providing genetic counseling for these families.
Notably, we only documented the following three novel variants, as they have not been previously reported in public databases, such as the Leiden Open Variation Database v.3.0 (LOVD,
In particular, the novel GCDH p.(Asn392Ter) variant was detected in trans with the pathogenic p.(Arg234Trp) variant in patient 3bINP-023. The p.(Arg234Trp) variant has been previously identified in homozygous state in two affected Polish sisters with a milder phenotype of glutaric acidemia, type I (OMIM #231670) [49]. In our patient, we observed a mild phenotype characterized by intellectual disability, seizures, abnormal movements, dyskinetic syndrome, truncal ataxia and dysmetria, abnormalities of the cerebral white matter and basal gray nuclei, along with malnutrition.
The LP variant p.(Pro489Arg) in GLUD1, which is responsible for autosomal dominant hyperinsulinism–hyperammonemia syndrome (OMIM #606762), was confirmed by Sanger sequencing in the heterozygous state in both patient 3bINP-085 and his father. After the identification of the variant in the apparently healthy father, a blood ammonia concentration test, which had a value of 216 ng/dL (reference range 31-123 ng/dL), was requested. This male patient, 3bINP-085, also presented co-occurrence with X-linked hemolytic anemia due to G6PD deficiency (OMIM #300908), which was attributed to the most prevalent worldwide G6PD haplotype, c.[202G>A;376A>G], or p.[Val68Met;Asn126Asp], conditioning G6PD deficiency [50].
The third novel BTD variant, p.(Cys451Ser), was present in a homozygous state in a five-year-old male patient (3bINP-090; Table 2) affected by autosomal recessive biotinidase deficiency (OMIM #253260), whose parents reported inbreeding and consanguinity. This patient exhibited mild intellectual disability, poor visual acuity, nerve optic atrophy, and pectus excavatum. As the missense p.(Cys451Ser) variant was classified as LP, we performed direct genotyping via Sanger sequencing on the parents, confirming their obligate carrier status and subsequently supporting its pathogenicity.
The co-occurrence of two monogenic traits was documented in 10.5% (N = 10/95) of our studied patients. This is quite similar to that reported (10.4%) in most studies included in a recent systematic review [28], although great variability has been noted in other series (nearly 5%) [51,52]. Most of these secondary findings are related to cardiovascular disease and hereditary cancer syndromes [28]; however, in some instances, G6PD deficiency is the second most frequently identified monogenic trait [51], as it is considered the most common enzymopathy among humans, affecting over 500 million people worldwide [50]. In our study population, 40% (N = 4/10; Table 4) of the overall identified secondary monogenic disorders were distributed among the three major disease categories reported.
The secondary actionable findings (i.e., cardiovascular disease or hereditary cancer syndromes) accounted for 3.15% (N = 3/95) of the overall study population (Table 4), which is in accordance with the 1-6% previously reported [10,53].
Finally, a treatment decision was taken after WES results in 24.2% (N = 23/95) of the studied patients. Changes in medical management were mainly related to the co-occurrence of a second monogenic disorder (N = 7/23). In fact, despite that in some patients the WES analysis results removed the suspicion of carrying an IEiM, it allowed the identification of other monogenic actionable disorders (i.e., patients 3bINP-001 and 3bINP-041), leading to a redirection of the medical management (Table 5). Changes in medical or nutritional management can be of different types, including redirection of care, initiation of new subspecialist care, changes in diet or medication, or major procedures, such as liver or kidney transplant [54]. In general, it is estimated that therapy guided by NGS results can reach 14.6% of the analyzed patients [28], although these proportions differ significantly among different studies, reaching 45.5% (N = 10/22) [9] to 52% in critically ill studied patients after WES analysis [10,54]. Importantly, the cohorts studied by the formerly mentioned authors were mainly composed of severely ill patients. In contrast, our cohort was entirely composed of metabolically compensated ambulatory patients who were previously diagnosed and treated according to their biochemical profile.
4. Materials and Methods
4.1. Study Population
An observational, descriptive, prospective, and cross-sectional study was conducted. Patients and their parents were contacted by telephone and invited to participate. The study included 95 unrelated Mexican mestizo individuals (51 females and 44 males) recruited from a cohort of pediatric patients (mean age 9 years) who attended the National Institute of Pediatrics, Mexico (
Patients were categorized into one of the following classes based on their biochemical phenotypes: amino acid, urea cycle, organic acid, carbohydrate, or lipid disorders. The included patients were assigned to two groups. Those bearing a well-defined biochemical phenotype that indicated a specific IEiM were assigned to Group 1, i.e., a high blood concentration of arginine was indicative of argininemia [56,57]. Group 2 included patients with either persistent or isolated nonspecific alterations in their amino acid and acylcarnitine profiles or with unexplained abnormalities in other laboratory studies, such as hypoglycemia and hyperammonemia.
4.2. Biochemical Testing and Phenotyping
In all cases, dried blood spot (DBS) samples, obtained from heel prick (in patients under 6 months of age) or finger prick (in patients above 6 months of age) via a standard protocol, were used to quantify amino acids, acylcarnitines, and succinylacetone quantification via tandem mass spectrometry using a conventional methodology previously described [58]. A plasma sample was also obtained for amino acid quantification via high-performance liquid chromatography (HPLC), and urinary organic acids were analyzed via gas chromatography coupled with mass spectrometry (GC/MS). In some cases, orotic acid was determined from urine. These determinations were made according to previously reported methodologies [58].
4.3. WES and Variant Analysis
Genomic DNA was extracted from DBS samples via the standard salting-out method. WES was performed via the xGen Exome Research Panel v2, either by itself or supplemented with the xGen human mtDNA panel and the xGen Custom Hyb Panel v1 (Integrated DNA Technologies, Coralville, Iowa, USA), and the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used to capture and sequence the protein-coding exons of ~20,000 known genes. The sequencing data were aligned to the GRCh37/hg19 human reference genome and the mitochondrial genome’s Revised Cambridge Reference Sequence (rCRS). Single-nucleotide variants (SNVs), small insertions and deletions (INDELs), copy number variants spanning at least 3 consecutive exons (CNVs), repeat expansion variants, and regions of homozygosity were called with open-source bioinformatics tools and in-house software, as previously described, and this was performed for positive, negative, and inconclusive patients [59]. Variant annotation, filtering, and classification were performed via EVIDENCE [59]. Common variants with allele frequencies > 5% in the gnomAD database [60] or >1% internally were filtered out, except for known pathogenic/likely pathogenic (P/LP) variants. Variant classification was performed based on the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines [16], along with the quantitative Bayesian scoring system [61]. All patients were subjected to clinical genetics assessment (A. G.-A., L. F.-H., and B. E.-O.) before their inclusion in this study. The patients’ clinical signs and symptoms were registered according to the Human Phenotype Ontology (HPO,
Patients were classified as positive when their genotype involved “pathogenic” or “likely pathogenic” variant(s) correlating with their phenotype and its inheritance mode. In contrast, inconclusive classification was determined when two variants of uncertain clinical significance (VUS) were identified or when only one P/LP variant partially explained a suspected or biochemically diagnosed autosomal recessive IEiM. Patients without any identifiable IEiM-associated variants were classified as negative. Co-occurrence was defined as the presence of a second monogenic entity in the same patient, including those expected findings according to the recorded clinical and biochemical phenotypes, and incidental or secondary findings. According to the ACMG, incidental findings are defined as results that are not related to the indication for ordering the sequencing but that may nonetheless be of medical value or utility [65,66]. On the other hand, secondary findings are defined as known pathogenic or expected pathogenic variants in a defined set of genes considered medically actionable, even when unrelated to the primary medical reason for testing [67]. Incidental and secondary findings were reported following the ACMG SF v.3.2. list [67]. Directed Sanger sequencing was performed on selected variants in 14 patients and, when available, on their parents to attempt to reclassify VUS as a “LP” or “P” variant by demonstrating the trans configuration, clarifying the mode of inheritance, or to identify clinically relevant parental genotypes for genetic counseling purposes. Based on this, genotypes were indicated according to the HGVS guidelines version 21.0.4 (
4.4. Statistical Analyses
Differences in the concordance between the biochemical initial diagnosis and WES results were investigated by the two-sided Fisher’s exact test in the general cohort, as well as between the two studied groups and between types of disorders. A p-value < 0.05 was considered to be statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). If applicable, descriptive statistics were applied. These statistical determinations were performed with GraphPad Prism version 10.1.1 (GraphPad Software, Boston, MA, USA).
4.5. Ethical Considerations
The Research, Ethics, and Biosafety Institutional Committees approved the protocol (institutional number 2022/051). Before carrying out the molecular study, the parents of the included patients signed an informed consent form, and those patients who could, due to their age and competence, granted their consent. Everyone was asked to decide whether they wanted to know about the secondary findings (ACMG SF v.3.2.) [67]. All the families received pre- and post-WES genetic counseling and medical follow-up.
5. Conclusions
In the 95 studied unrelated Mexican pediatric patients included in this study, the previous specific biochemical diagnosis of an IEiM correlated with a higher genetic concordance of WES (91.3%, N = 63/69 patients) compared with unspecific biochemical alterations suggestive of these disorders (23.1%, N = 6/26 patients). The overall diagnostic concordance between the initial biochemical profile indicating an IEiM diagnosis and the responsible genotype identified through WES was 72.6% (N = 69/95 patients). These results highlight the importance of biochemical studies as a first-tier diagnostic approach in all patients with suspected IiEM to achieve prompt and specific implementation of therapeutic management, as well as to increase the overall diagnostic yield of WES. The identified underlying genotypic IEiM spectrum involved 83 pathogenic, likely pathogenic, and VUS variants, including three novel ones in GLUD1, BTD, and GCDH, which were distributed among 29 different genes responsible for amino acid, organic acid, urea cycle, carbohydrate, and lipid disorders. Unsolved WES results were identified in 27.4% (N = 26/95) of the patients. The proportion of patients with a second monogenic disease (10.5%) was similar to that reported in the literature (10.4%). The second monogenic diseases found were mainly cardiovascular, hereditary cancer syndromes, and G6PD deficiency. WES-directed modifications in medical or nutritional management were performed in 33.6% (N = 32/95) of patients. In 56.2% of them (N = 18/32), the changes were attributed to discordance between the initial and final diagnosis, the co-occurrence of a second monogenic trait or syndromic entities unrelated to IEiM, or an initial unspecific diagnosis with a negative WES result.
M.V.-A., M.A.A.-O., A.G.-d.A., L.F.-H., M.E.R.-F. and B.E.-O. contributed equally to this work. Conceptualization, M.V.-A., C.F.-L. and M.A.A.-O.; methodology, S.-W.R., H.L., M.A.A.-O., M.E.R.-F., B.E.-O., L.F.-H. and A.G.-d.A.; variant interpretation and reclassification analysis, M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A., S.-W.R. and H.L.; clinical and nutritional follow-up, L.B.-M., R.I.C.-N., S.G.-L. and L.L.-M.; software, S.-W.R., H.L., M.E.R.-F., L.F.-H., M.A.A.-O. and I.I.-G.; validation, M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A. and M.A.A.-O.; formal analysis, M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A., M.V.-A., C.F.-L. and M.A.A.-O.; investigation, S.G.-L., L.L.-M., L.B.-M. and R.I.C.-N.; resources, M.V.-A., C.F.-L. and M.A.A.-O.; data curation, S.-W.R., H.L., I.I.-G., M.A.A.-O., B.E.-O. and A.G.-d.A.; writing—original draft preparation, M.V.-A., C.F.-L., M.A.A.-O., M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A., S.-W.R. and H.L.; writing—review and editing, M.V.-A., C.F.-L., M.A.A.-O., M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A., L.B.-M., R.I.C.-N., S.G.-L., L.L.-M., I.I.-G., S.-W.R., H.L. and on behalf of RaDiMEG; visualization, M.E.R.-F., B.E.-O., L.F.-H., A.G.-d.A. and C.F.-L.; supervision, M.V.-A., C.F.-L. and M.A.A.-O.; project administration, M.V.-A. and C.F.-L.; funding acquisition, M.V.-A., C.F.-L. and M.A.A.-O. All authors have read and agreed to the published version of the manuscript.
This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (Ethics, Research, and Biosafety Committees) of the National Institute of Pediatrics (reference number 2022/051, approval date 12 August 2022).
Written informed consent was obtained from the patients involved in the study or by their parents for the publication of this paper.
Publicly available datasets were analyzed in this study. The data can be found here: ClinVar:
We thank the patients and their families for their support and commitment. The authors gratefully acknowledge Aida Janette Hernández-Montiel, Luis Ricardo Morales-González, and Jaime Torres-Marcial for their technical assistance.
Authors Seung Woo Ryu and Hane Lee were employed by the company 3billion, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare no conflicts of interest.
Footnotes
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Gene variants responsible for IEiM identified in Mexican patients, categorized by pathogenicity.
| Type of Disorder | Disease | Gene | Total Number of Variants | Pathogenic | Likely Pathogenic | Variant of Uncertain Significance |
|---|---|---|---|---|---|---|
| Amino acid disorders | Maple syrup urine disease (MSUD) type Ib (OMIM #620698) | BCKDHB | 6 | 4 | 2 | 0 |
| MSUD, type II (OMIM #620699) | DBT | 6 | 3 | 1 | 2 | |
| MSUD, type Ia (OMIM #248600) | BCKDHA | 2 | 1 | 1 | 0 | |
| Homocystinuria, B6-responsive and nonresponsive types (OMIM #236200) | CBS | 2 | 2 | 0 | 0 | |
| Cystinosis, nephropathic (OMIM #219800) | CTNS | 2 | 0 | 2 | 0 | |
| Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (OMIM #238970) | SLC25A15 | 1 | 1 | 0 | 0 | |
| Organic acid disorders | HMG-CoA lyase deficiency, 3-OH-3-methylglutaric acidemia (OMIM #246450) | HMGCL | 7 | 5 | 2 | 0 |
| Glutaric Acidemia Type 1 (OMIM #231670) | GCDH | 5 | 5 | 0 | 0 | |
| Isovaleric acidemia (OMIM #243500) | IVD | 5 | 4 | 1 | 0 | |
| Biotinidase deficiency (OMIM #253260) | BTD | 4 | 3 | 1 | 0 | |
| Mitochondrial DNA depletion syndrome 9 (encephalomyopathic type with methylmalonic aciduria) (OMIM #245400) | SUCLG1 | 2 | 1 | 1 | 0 | |
| Beta-ketothiolase deficiency or mitochondrial acetoacetyl-CoA thiolase deficiency or alphamethylacetoacetic aciduria (OMIM #203750) | ACAT1 | 3 | 1 | 2 | 0 | |
| Holocarboxylase synthetase deficiency (OMIM #253270) | HLCS | 1 | 1 | 0 | 0 | |
| 3-Methylcrotonyl-CoA carboxylase 2 deficiency (OMIM #210210) | MCCC2 | 1 | 0 | 1 | 0 | |
| Succinyl CoA:3-oxoacid CoA transferase deficiency (OMIM #245050) | OXCT1 | 1 | 1 | 0 | 0 | |
| Urea cycle disorders | Argininemia (OMIM #207800) | ARG1 | 8 | 5 | 3 | 0 |
| Citrullinemia (OMIM #215700) | ASS1 | 6 | 4 | 2 | 0 | |
| Ornithine transcarbamylase deficiency (OMIM #311250) | OTC | 2 | 2 | 0 | 0 | |
| Argininosuccinic aciduria (OMIM #207900) | ASL | 1 | 0 | 1 | 0 | |
| Carbohydrate disorders | Glycogen storage disease Ia (OMIM #232200) | G6PC1 | 5 | 4 | 1 | 0 |
| Glycogen storage disease IIIb (OMIM #232400) | AGL | 1 | 0 | 1 | 0 | |
| Glycogen storage disease Ib (OMIM: 232220) | SLC37A4 | 2 | 1 | 1 | 0 | |
| Diarrhea type 4, malabsorptive, congenital (OMIM #610370) | NEUROG3 | 1 | 1 | 0 | 0 | |
| Galactokinase deficiency with cataracts (OMIM #230200) | GALK1 | 2 | 0 | 0 | 2 | |
| Hyperinsulinemic hypoglycemia, familial, type 1 (OMIM #256450) | ABCC8 | 1 | 1 | 0 | 0 | |
| Hyperinsulinism-hyperammonemia syndrome (OMIM #606762) | GLUD1 | 1 | 0 | 1 | 0 | |
| Hyperinsulinemic hypoglycemia, familial, type 2 (OMIM #601820) | KCNJ11 | 1 | 0 | 1 | 0 | |
| Lipid disorders | Acyl-CoA dehydrogenase, medium chain deficiency (MCAD, OMIM #201450) | ACADM | 3 | 3 | 0 | 0 |
| Hypercholesterolemia familial type 1 (OMIM #143890) | LDLR | 1 | 1 | 0 | 0 | |
| Total | 83 | 54 | 25 | 4 |
Genotypic spectrum underlying IEiM in positive cases of the studied population.
| Amino Acid Disorders | ||||||||
|---|---|---|---|---|---|---|---|---|
| Biochemical Phenotype | Initialy Suspected Disease | Responsible Gene (Reference Sequence) | Patient ID | LOVD Individual Accession Number | Genotype A | Protein Change | Final Diagnosis | Inheritance |
| Elevated circulating branched chain amino acid concentration (HP:0008344) | MSUD | BCKDHB (NM_183050.4) | 3bINP-066 | 451632 | c.[152del];[152del] | p.[Val51GlyfsTer21];[Val51GlyfsTer21] | MSUD type Ib (OMIM #620698) | AR |
| 3bINP-080 | 451644 | c.[564T>A];[564T>A] | p.[Cys188Ter];[Cys188Ter] | |||||
| 3bINP-020 | 451365 | c.564T>A(;)1087T>A | p.(Cys188Ter)(;)(Tyr363Asn) | |||||
| 3bINP-077 | 451640 | c.853C>T(;)667G>C | p.(Arg285Ter)(;)(Gly223Arg) | |||||
| 3bINP-004 | 450321 | c.[970C>T];[970C>T] | p.[Arg324Ter];[Arg324Ter] | |||||
| 3bINP-013 | 450471 | c.[1087T>A];[1087T>A] | p.[Tyr363Asn];[Tyr363Asn] | |||||
| DBT (NM_001918.5) | 3bINP-069 | 451637 | c.[75_76del];[75_76del] B | p.[Cys26TrpfsTer2];[Cys26TrpfsTer2] | MSUD type II (OMIM #620699) | |||
| 3bINP-092 | 451652 | |||||||
| 3bINP-104 | 451662 | c.[263_265del];[263_265del] | p.[Glu88del];[Glu88del] | |||||
| 3bINP-027 | 451439 | c.[434-15_434-4del];[434-15_434-4del] | p.[?];[?] | |||||
| 3bINP-081 | 451645 | c.670G>T(;)434-15_434-4del | p.(Glu224Ter)(;)(?) | |||||
| BCKDHA (NM_000709.4) | 3bINP-062 | 451630 | c.890G>A(;)1192G>T | p.(Arg297His)(;)(Glu398Ter) | MSUD type Ia (OMIM #248600) | |||
| Hyperammonemia (HP:0001987) | Gyrate atrophy or HHH Sx | SLC25A15 (NM_014252.4) | 3bINP-021 | 451367 | c.[113_116dup];[113_116dup] | p.[Phe40AspfsTer4];[Phe40AspfsTer4] | Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (OMIM #238970) | AR |
| Homocystinuria (HP:0002156) | Homocystinuria | CBS | 3bINP-046 | 451595 | c.[572C>T];[572C>T] C | p.[Thr191Met];[Thr191Met] | Homocystinuria (OMIM #236200) | AR |
| 3bINP-087 | 451647 | |||||||
| 3bINP-109 | 451663 | |||||||
| 3bINP-049 | 451597 | c.[1126G>A];[1126G>A] | p.[Asp376Asn];[Asp376Asn] | |||||
| Corneal crystals (HP: 0000531), Fanconi syndrome (HP: 0011463) C | Cystinosis | CTNS | 3bINP-082 | 451648 | c.[22_23del];[1036_1047del] D | p.[Ile8PhefsTer13];[Asp346_Phe349del] | Cystinosis, nephropathic (OMIM #219800) | AR |
| Organic acid disorders | ||||||||
| Biochemical phenotype | Initialy suspected disease | Responsible gene | Patient ID | LOVD individual accession number | Genotype A | Protein change | Final diagnosis | Inheritance |
| Decreased circulating biotinidase concentration (HP:0410145) | Biotinidase deficiency | BTD | 3bINP-054 | 451617 | c.468G>T(;)1270G>C | p.(Lys156Asn)(;)(Asp424His) | Biotinidase deficiency(OMIM #253260) | AR |
| Biotinidase deficiency with atypical outcome, epilepsy | 3bINP-101 | 451656 | c.[754T>G];[754T>G] | p.[Trp252Gly];[Trp252Gly] | ||||
| Biotinidase deficiency | 3bINP-053 | 451616 | c.1270G>C(;)754T>G | p.(Asp424His)(;)(Trp252Gly) | ||||
| Biotinidase deficiency | 3bINP-011 | 450470 | c.[1270G>C];[1270G>C] | p.[Asp424His];[Asp424His] | ||||
| Elevated circulating acylcarnitine concentration (HP: 0045045), organic aciduria (HP:0001992) | Multiple carboxylase deficiency | 3bINP-090 | 451651 | c.[1352G>C];[1352G>C] | p.[Cys451Ser];[Cys451Ser] | |||
| Organic aciduria (HP:0001992) | Multiple carboxylase deficiency | SUCLG1 | 3bINP-028 | 451440 | c.40A>T(;)548T>C | p.(Met14Leu)(;)(Ile183Thr) | Succinate-CoA ligase, alpha subunit deficiency (OMIM #245400) | AR |
| Elevated circulating acylcarnitine concentration (HP:0045045) | Multiple carboxylase deficiency | 3bINP-084 | 451646 | |||||
| Increased circulating isovaleric acid concentration (HP:0033148) | Isovaleric acidemia | IVD | 3bINP-057 | 451600 | c.[149G>C];[149G>C] | p.[Arg50Pro];[Arg50Pro] | Isovaleric acidemia (OMIM #243500) | AR |
| 3bINP-019 | 451363 | c.[850C>T];[850C>T] | p.[Arg284Trp];[Arg284Trp] | |||||
| 3bINP-003 | 450320 | c.[1065G>C];[1065G>C] | p.[Lys355Asn];[Lys355Asn] | |||||
| 3bINP-079 | 451643 | c.[1175G>A];[1175G>A] | p.[Arg392His];[Arg392His] | |||||
| 3bINP-005 | 450322 | g.[(?_40710329)_(40710462_?)del];[(?_40710329)_(40710462_?)del](homozygous exon 12 deletion) | p.[?];[?] | |||||
| 3-methylglutaric aciduria (HP:0410051) | 3-hydroxy-3-methylglutaryl-CoA lyase deficiency | HMGCL | 3bINP-042 | 451460 | c.[109G>T];[109G>T] | p.[Glu37Ter];[Glu37Ter] | 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (OMIM #246450) | AR |
| 3bINP-035 | 451457 | c.[112G>T];[112G>T] | p.[Val38Phe];[Val38Phe] | |||||
| 3bINP-010 | 450325 | c.121C>T(;)233C>T | p.(Arg41Ter)(;)(Ser78Phe) | |||||
| 3bINP-074 | 451639 | c.230del(;)31C>T | p.(Val77GlyfsTer16)(;)(Arg11Ter) | |||||
| 3bINP-032 | 451445 | c.[505_506del];[505_506del] | p.[Ser169LeufsTer8];[Ser169LeufsTer8] | |||||
| Concentration of glutaric acid in the urine above the upper limit of normal (HP:0003150) | Glutaric aciduria | GCDH | 3bINP-018 | 451362 | c.263G>A(;)1204C>T | p.(Arg88His)(;)(Arg402Trp) | Glutaric aciduria (OMIM #231670) | AR |
| 3bINP-023 | 451436 | c.[700C>T];[1173_1174insT] G | p.[Arg234Trp];[Asn392Ter] | |||||
| 3bINP-068 | 451635 | c.[1082+31_1243+678del];[1082+31_1243+678del](homozygous whole exon 11 deletion) | p.[Ala362Tyrfs*3];[Ala362TyrfsTer3] | |||||
| Elevated circulating acylcarnitine concentration (HP:0045045) | Organic aciduria | ACAT1 | 3bINP-047 | 451615 | c.[473A>G];[473A>G] | p.[Asn158Ser];[Asn158Ser] | Alpha-methylacetoacetic aciduria (OMIM #203750) | AR |
| Organic aciduria (HP:0001992) | Organic aciduria | 3bINP-017 | 450485 | c.826+3_826+6del(;)200T>G | p.(?)(;)(Leu67Arg) | |||
| Organic aciduria (HP:0001992) | 3-Methylcrotonyl-CoA carboxylase deficiency | MCCC2 | 3bINP-059 | 451602 | c.[1356G>A];[1356G>A] | p.[Met452Ile];[Met452Ile] | 3-Methylcrotonyl-CoA carboxylase deficiency (OMIM #210210) | AR |
| Ketosis (HP:0001946) | Ketone bodies defect | OXCT1 | 3bINP-060 | 451603 | c.[1243del];[1243del] | p.[Ile415TyrfsTer6];[Ile415TyrfsTer6] | Succinyl-CoA:3-oxoacid- CoA transferase deficiency (OMIM #245050) | AR |
| Urea cycle disorders | ||||||||
| Biochemical phenotype | Initialy suspected disease | Responsible gene | Patient ID | LOVD individual accession number | Genotype A | Protein change | Final diagnosis | Inheritance |
| Hyperargininemia (HP:0500153) | Argininemia | ARG1 | 3bINP-033 | 451456 | c.3G>A(;)767_769del | p.(Met1?)(;)(Glu256del) | Argininemia (OMIM #207800) | AR |
| 3bINP-067 | 451634 | c.61C>T(;)466-1G>C E | p.(Arg21Ter)(;)(?) | |||||
| 3bINP-078 | 451642 | |||||||
| 3bINP-037 | 451458 | c.61C>T(;)892G>C | p.(Arg21Ter)(;)(Ala298Pro) | |||||
| 3bINP-055 | 451599 | c.[425G>A];[425G>A] | p.[Gly142Glu];[Gly142Glu] | |||||
| 3bINP-073 | 451638 | c.466-1G>C(;)787G>T | p.(?)(;)(Glu263Ter) | |||||
| 3bINP-105 | 451658 | c.425G>A(;)871C>T | p.(Gly142Glu)(;)(Arg291Ter) | |||||
| Elevated plasma citrulline (HP:0011966) | Citrullinemia | ASS1 | 3bINP-022 | 451368 | c.[34A>G];[34A>G] | p.[Ser12Gly];[Ser12Gly] | Citrullinemia (OMIM #215700) | AR |
| 3bINP-106 | 451659 | c.[256C>T];[256C>T] | p.[Arg86Cys];[Arg86Cys] | |||||
| 3bINP-008 | 450323 | c.256C>T(;)836G>A F | p.(Arg86Cys)(;)(Arg279GIn) | |||||
| 3bINP-065 | 451631 | |||||||
| 3bINP-095 | 451654 | c.256C>T(;)1194-19_1197dup | p.(Arg86Cys)(;)(?) | |||||
| 3bINP-015 | 450482 | c.970G>A(;)40G>A | p.Gly324Ser)(;)(Gly14Ser) | |||||
| Orotic aciduria (HP:0003218) | Ornithine transcarbamylase deficiency | OTC | 3bINP-043 | 451463 | c.[583G>A];[583=] (heterozygous female) | p.[Gly195Arg];[Gly=] (heterozygous female) | Ornithine transcarbamylase deficiency (OMIM #311250) | X-linked |
| 3bINP-048 | 451596 | c.[803T>C];[0] (hemizygous male) | p.[Met268Thr];[0] (hemizygous male) | |||||
| Argininosuccinic aciduria (HP:0025630) | Argininosuccinic aciduria | ASL | 3bINP-014 | 450472 | c.[209T>C];[209T>C] | p.[Val70Ala];[Val70Ala] | Argininosuccinic aciduria (OMIM #207900) | AR |
| Carbohydrate disorders | ||||||||
| Biochemical phenotype | Initialy suspected disease | Responsible gene | Patient ID | LOVD individual accession number | Genotype A | Protein change | Final diagnosis | Inheritance |
| Abnormal hepatic glycogen storage (HP:0500030), hypoglycemia (HP:0001943) | Glycogen storage disease type I | G6PC1 | 3bINP-029 | 451441 | c.379_380dup(;)1039C>T | p.(Tyr128ThrfsTer3)(;)(Gln347Ter) | Glycogen storage disease Ia (OMIM #232200) | AR |
| Hepatomegaly (HP:0002240), hypoglycemia (HP:0001943), hypertriglyceridemia (HP:0002155), hypercholesterolemia (HP:0003124), hepatic steatosis (HP:0001397), abnormal hepatic glycogen storage (HP:0500030) | 3bINP-107 | 451660 | c.[533C>T];[500G>A] | p.[Pro178Leu];[Cys167Tyr] | ||||
| Hepatomegaly (HP:0002240), hypoglycemia (HP:0001943) | 3bINP-102 | 451657 | c.[809G>T];[809G>T] | p.[Gly270Val];[Gly270Val] | ||||
| Abnormal hepatic glycogen storage (HP:0500030), hypoglycemia (HP:0001943) | Glycogen storage disease type III | AGL | 3bINP-025 | 451437 | c.[2803G>T];[2803G>T] | p.[Gly935Cys];[Gly935Cys] | Glycogen storage disease IIIb (OMIM #232400) | AR |
| Abnormal hepatic glycogen storage (HP:0500030), hypoglycemia (HP:0001943), neutropenia (HP: 0001875) | Glycogen storage disease Ib | SLC37A4 | 3bINP-097 | 451655 | c.82C>T(;)1130G>A | p.(Arg28Cys)(;)(Gly377Asp) | Glycogen storage disease Ib (OMIM #232220) | AR |
| Type 1 diabetes mellitus (HP:0100651) | Mauriac syndrome, type 1 diabetes mellitus | NEUROG3 | 3bINP-100 | 451661 | c.[117del];[117del] | p.[Thr40LeufsTer38];[Thr40LeufsTer38] | Diarrhea type 4, malabsorptive, congenital (OMIM #610370) | AR |
| Hypoglycemia (HP:0001943) | Hypoglycemia | ABCC8 | 3bINP-045 | 451461 | c.[2506C>T];[2506=] H | p.[Arg836Ter];[Arg=] | Hyperinsulinemic hypoglycemia, familial, type 1 (OMIM #256450) | AD |
| Hypoglycemia, abnormal circulating glucose-6-phosphate dehydrogenase concentration (HP:0001943, HP:0410176) | Hypoglycemia and glucose-6-phosphate dehydrogenase deficiency | GLUD1 | 3bINP-085 | 451650 | c.[1466C>G];[1466=] | p.[Pro489Arg];[Pro=] | Hyperinsulinism-hyperammonemia syndrome (OMIM #606762) | AD |
| Hypoglycemia (HP:0001943) | Hypoglycemia | KCNJ11 | 3bINP-030 | 451442 | c.[560C>T];[560=] H | p.[Ala187Val];[Ala=] | Hyperinsulinemic hypoglycemia, familial, type 2 (OMIM #601820) | AD |
| Lipid defects | ||||||||
| Biochemical phenotype | Initialy suspected disease | Responsible gene | Patient ID | LOVD individual accession number | Genotype A | Protein change | Final diagnosis | Inheritance |
| Elevated circulating acylcarnitine concentration (HP:0045045) | Acyl-CoA dehydrogenase deficiency (MCAD) | ACADM | 3bINP-044 | 451464 | c.799G>A(;)959C>A | p.(Gly267Arg)(;)(Ser320Ter) | MCAD (OMIM #201450) | AR |
| Hypercholesterolemia (HP:0003124) | Familial hypercholesterolemia | LDLR | 3bINP-058 | 451601 | c.[337dup];[337=] | p.[Glu113GlyfsTer17];[Glu=] | Familial hypercholesterolemia type 1 (OMIM #143890) | AD |
A Described genotypes considered only pathogenic and likely pathogenic variants (see
Biochemical primary biomarkers related to unsolved cases (inconclusive and negative) in the studied population.
| Study Group | Patient ID | HPO | Biomarker | Concentration (Reference Value) | Suspected Disease | Gene (Reference Sequence, Encoded Protein) | Variant 1 (Classification) | Variant 2 (Classification) | Conclusion |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 3bINP-036 | 0045045 | 3-hydroxy-isovalerylcanitine + methylmalonylcarnitine | 1.36 μmol/L (0.83) | Organic acidemia | HLCS (NM_001352514.2, Holocarboxylase synthetase) | c.2361_2362insT or p.(Val788CysfsTer108) (pathogenic) | Not identified | Inconclusive A |
| 3bINP-052 | 0045045 | Hexanoylcarnitine | 0.16 μmol/L (0.12) | Medium chain acyl-CoA dehydrogenase deficiency | ACADM (NM_000016.6, Medium chain acyl-CoA dehydrogenase) | c.985A>G or p.(Lys329Glu) (pathogenic) | Not identified | Inconclusive B | |
| Octanoylcarnitine | 0.36 μmol/L (0.16) | ||||||||
| Decanoylcarnitine | 0.37 μmol/L (0.21) | ||||||||
| 3bINP-072 | 0500030 | Glycogen | Positive liver biopsy | Glycogen storage disease | Not identified | Not identified | Not identified | Negative | |
| 3bINP-076 | 0008344 | Leucine + isoleucine | 1924 μmol/L (40–228) | Maple syrup urine disease | DBT (NM_001918.5, Dihydrolipoamide branched-chain transacylase) | c.1210-3T>A or p.(?) (VUS) | c.1210-3T>A or p.(?) (VUS) | Inconclusive | |
| Valine | 443 μmol/L (37–237) | ||||||||
| Alloisoleucine | Not determined | ||||||||
| 0001992 | Urinay organic acid profile | Elevated excretion of branched chain keto acids | |||||||
| 3bINP-089 | 0012024 | Galactose (with Galactose-1-P uridyltransferase normal activity) | 19.99 mg/dL (<12) | Galactosemia | GALK1 (NM_000154.2, Galctose kinase) | c.56C>A or p.(Ala19Asp) (VUS) | c.182C>T or p.(Thr61Met) (VUS) | Inconclusive | |
| 3bINP-094 | 0008344 | Leucine + isoleucine | 3226 μmol/L (<253) | Maple syrup urine disease | DBT (NM_001918.5, Dihydrolipoamide branched-chain transacylase) | c.1261G>T or p.(Gly421Trp) (VUS) | c.1261G>T or p.(Gly421Trp) (VUS) | Inconclusive | |
| Valine | 1286 μmol/L (<282) | ||||||||
| Alloisoleucine | 64 μmol/L (Not detectable) | ||||||||
| 0001992 | Urinay organic acid profile | Elevated excretion of branched chain keto acids | |||||||
| 2 | 3bINP-001 | 0004359 | Propionylcarnitine | 4.6 μmol/L (<2.5) | Organic acidemia | Not identified | Not identified | Not identified | Negative |
| 3bINP-006 | 0045045 | Tetradecanoylcarnitine | 0.33 μmol/L (<0.31) | Organic acidemia | Not identified | Not identified | Not identified | Negative | |
| 0001992 | Urinay organic acid profile | Elevated excretion of adipic, suberic and sebasic acids | |||||||
| 3bINP-007 | 0008358 | Proline | 393 μmol/L (<290) | Hyperprolinemia | Not identified | Not identified | Not identified | Negative | |
| 3bINP-009 | 0001943 | Glucose | <40 mg/dL (70) | Carbohydrate disorder | Not identified | Not identified | Not identified | Negative | |
| 0000842 | Hyperinsulinemia | 19.3 uU/mL (<2) | |||||||
| 3bINP-012 | 0001943 | Glucose | <40 mg/dL (70) | Carbohydrate disorder | Not identified | Not identified | Not identified | Inconclusive C | |
| 0000842 | Hyperinsulinemia | 26.8 uU/mL (<2) | |||||||
| 3bINP-016 | 0003235 | Methionine | 99 μmol/L (9–42) | Hypermethioninemia | Not identified | Not identified | Not identified | Negative | |
| 0002156 | Homocysteine | 8 μmol/L (0–6.4) | |||||||
| 3bINP-024 | 0001987 | Hyperammonemia | 117 μmol/L (9–35) | Urea cycle disorder | Not identified | Not identified | Not identified | Negative | |
| 3bINP-026 | 0003348 | Alanine | 1007 μmol/L (<605) | Hyperalaninemia | Not identified | Not identified | Not identified | Negative | |
| 3bINP-031 | 0003235 | Methionine | 209 μmol/L (<52), | Hypermethioninemia | Not identified | Not identified | Not identified | Negative | |
| Met/Phe ratio | Met/Phe 4.2 (<1.4) | ||||||||
| 3bINP-034 | 0045045 | Hexadecanoylcarnitine | 2.98 μmol/L (<2.4) | Fatty acid oxidation defect | Not identified | Not identified | Not identified | Negative | |
| Octadecenoylcarnitine | 2.45 μmol/L (<1.6) | ||||||||
| 3-hydroxy-octadecenoylcarnitine | 0.06 μmol/L (<0.03) | ||||||||
| Octadecadienoylcarnitine | 0.69 μmol/L (<0.47) | ||||||||
| 3bINP-038 | 0008344 | Leucine + isoleucine | 426 (<253) | Maple syrup urine disease | Not identified | Not identified | Not identified | Negative | |
| Valine | 446 μmol/L (<282) | ||||||||
| Xleu (Leu + Ile)/Phe ratio | 7.12 (<3.95) | ||||||||
| Xleu (Leu + Ile)/Ala ratio | 10.7 (<0.43) | ||||||||
| Val/Phe ratio | 7.42 (<4.95) | ||||||||
| 3bINP-039 | 0008358 | Proline | 338 μmol/L (<290) | Hyperprolinemia | Not identified | Not identified | Not identified | Negative | |
| 3bINP-041 | 0012556 | beta-Alanine | 8 μmol/L (<5) | Hyperbeta-alaninemia | Not identified | Not identified | Not identified | Negative | |
| 0020079 | beta-Alaninuria | 256 mmol/mol creatinine (<6) | |||||||
| 0500138 | Serine | 194 μmol/L (85-185) | |||||||
| 0002154 | Glycine | 356 μmol/L (138-349) | |||||||
| 3bINP-050 | 0001992 | Propionylcarnitine | 10.7 μmol/L (<4.3) | Organic acidemia | Not identified | Not identified | Not identified | Negative | |
| 3bINP-051 | 0045045 | Free carnitine | 134 μmol/L (<53) | Fatty acid oxidation defect | Not identified | Not identified | Not identified | Negative | |
| Propionylcarnitine | 29 μmol/L (<4.2) | ||||||||
| Butyrylcarnitine | 1.5 μmol/L (<0.5) | ||||||||
| Hexadecanoylcarnitine | 5.1 μmol/L (<2.2) | ||||||||
| Tetradecanoylcarnitine | 0.41 μmol/L (<0.19) | ||||||||
| Octadecanoylcarnitine | 2.7 μmol/L (<0.87) | ||||||||
| Octadecenoylcarnitine | 6.5 μmol/L (<2.8) | ||||||||
| 3bINP-056 | 0045045 | Free carnitine | 456 μmol/L (<87) | Fatty acid oxidation defect | Not identified | Not identified | Not identified | Negative | |
| Hexadecanoylcarnitine | 0.32 μmol/L (<0.23) | ||||||||
| Octadecanoylcarnitine | 0.13 μmol/L (<0.1) | ||||||||
| Free carnitine/(hexadecanoylcarnitine + octadecanoylcarnitine) ratio | 1013 (<69) | ||||||||
| 3bINP-063 | 0001992 | Urinay organic acid profile | Elevated excretion of 3-hydroxybutiric and acetoacetic acids | Organic acidemia | Not identified | Not identified | Not identified | Negative | |
| 3bINP-070 | 0008344 | Leucine + isoleucine | 265 μmol/L (<253) | Maple syrup urine disease | Not identified | Not identified | Not identified | Negative | |
| Valine | 303 μmol/L (<282) | ||||||||
| 0008358 | Proline | 439 μmol/L (<290) | |||||||
| 3bINP-093 | 0008344 | Leucine + isoleucine | 349 μmol/L (<253) | Organic acidemia | Not identified | Not identified | Not identified | Negative | |
| Valine | 345 μmol/L(<282) | ||||||||
| 0045045 | Butyrylcarnitine | 0.52 μmol/L (<0.45) | |||||||
| 3bINP-103 | 0001992 | Urinay organic acid profile | Elevation of 2-hydroxybutiric and 3-OH butyric acid | Organic acidemia | Not identified | Not identified | Not identified | Negative | |
| 0001942 | Hyperlactatemia | 5.8 (1-3.3 mmol/L) | |||||||
| 0045045 | 3-hydroxy-isovalerylcanitine + methylmalonylcarnitine | 1.11 μmol/L (<0.83) |
A Patient with a normal chromosomal microarray analysis result. B No other methodology was applied to the identification of the second pathogenic allele. C A possible androgenetic/biparental chimerism or genome-wide paternal uniparental disomy is still under study.
Co-occurrence of two monogenic diseases due to expected, incidental, or secondary findings in the studied patients.
| Study Group | Patient ID | HPO | Observed Biochemical Abnormality | 1st Disease Detected | Gene Responsible of First Disease | Genotype A | Identified Second Monogenic Disease | Gene Responsible of Second Disease | Genotype A | Type of Finding |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 3bINP-021 | 12026 | Hyperornithinemia | Hyperornithinemia-hyperammonemia-hyperhomocitrullinuria syndrome (OMIM #238970) | SLC25A15 | NM_014252.4:c.[113_116dup];[113_116dup] or p.[Phe40AspfsTer4];[Phe40AspfsTer4] | Autosomal dominant polydactyly, postaxial, types A1 and B (OMIM #174200) | GLI3 | NM_000168.6:c.[3740_3743dup];[3740=] or p.[Cys1249AlafsTer3];[Cys=] | Expected |
| 0001987 | Hyperammonemia | |||||||||
| 3bINP-054 | 0001992 | Biotinidase deficiency | Biotinidase deficiency (OMIM #253260) | BTD | NM_001370658.1:c.468G>T(;)1270G>C or p.(Lys156Asn)(;)(Asp424His) | Autosomal dominant FGFR2-related disorder (OMIM *176943) | FGFR2 | NM_000141.5:c.[923A>G];[923=] or p.[Tyr308Cys];[Tyr=] | Expected | |
| 3bINP-069 | 0008344 | Elevated circulating branched chain amino acid concentration | MSUD type II (OMIM #620699) | DBT | NM_001918.5:c.[75_76del];[75_76del] or p.[Cys26TrpfsTer2];[Cys26TrpfsTer2] | Autosomal recessive ATP-binding cassette, subfamily a, member 4 (ABCA4)-related disorder (OMIM *601691) | ABCA4 | NM_000350.3:c.[2453G>A];[2453G>A] or p.[Gly818Glu];[Gly818Glu] | Incidental | |
| 3bINP-074 | 0410051 | 3-methylglutaric aciduria | HMG-CoA lyase deficiency (OMIM #246450) | HMGCL | NM_000191.3:c.230del(;)31C>T or p.(Val77GlyfsTer16)(;)(Arg11Ter) | Autosomal dominant Lynch syndrome (OMIM #614350) | MSH6 | NM_000179.3:c.[2150_2153del];[2150=] or p.[Val717AlafsTer18];[Val=] | Secondary | |
| 3bINP-082 | 0000531 | Cystinosis | Nephropathic cystinosis (OMIM #219800) | CTNS | NM_004937.3:c.[22_23del];[1036_1047del] or p.[Ile8PhefsTer13];[Asp346_Phe349del] | X-linked Alport syndrome type 1 (OMIM #301050) | COL4A5 | NM_033380.3:c.[3088G>A];[3088=] or p.[Gly1030Ser];[Gly=] | Expected | |
| 3bINP-109 | 0002156 | Homocystinuria | Homocystinuria, B6-responsive and nonresponsive types (OMIM #236200) | CBS | NM_000071.3:c.[572C>T];[572C>T] or p.[Thr191Met];[Thr191Met] | Autosomal dominant Fleck corneal dystrophy (OMIM #121850) | PIKFYVE | NM_015040.4:c.[853_854del];[853=] or p.[Leu285PhefsTer19];[Leu=] | Incidental | |
| 2 | 3bINP-045 | 0001943 | Hypoglycemia | Autosomal dominant form of Hyperinsulinemic hypoglycemia familial type 1 (OMIM #256450) | ABCC8 | NM_000352.6:c.[2506C>T];[2506=] or p.[Arg836Ter];[Arg=] | Autosomal dominant RET-related disorders, including Multiple endocrine neoplasia (MEN) IIA (OMIM #171400), MEN IIB (OMIM #162300), and familial medullary thyroid carcinoma (OMIM #155240) | RET | NM_020975.6:c.[2410G>A];[2410=] or p.[Val804Met];[Val=] | Secondary |
| 3bINP-047 | 0045045 | Inespecific acylcarnitine alterations | Alpha-methylacetoacetic aciduria (OMIM #203750) | ACAT1 | NM_000019.4:c.[473A>G];[473A>G] or p.[Asn158Ser];[Asn158Ser] | Autosomal dominant Cardiomyopathy, dilated, type 1G (OMIM #604145) | TTN | NM_001267550.2:c.[87470_87471del];[87470=] or p.[Leu29157GlnfsTer6];[Leu=] | Secondary | |
| 3bINP-085 | 0001943 | Hypoglycemia | Autosomal dominant form of Hyperisulinism-hyperammonemia syndrome (OMIM #606762) | GLUD1 | NM_005271.5:c.[1466C>G];[1466=] orp.[Pro489Arg];[Pro=] | X-linked Glucose-6-phosphate dehydrogenase deficiency (OMIM #300908) | G6PD | Hemizygous male for haplotype NM_001360016.2:c.[376A>G;202G>A];[0] or p.[Asn126Asp;Val68Met];[0] | Expected | |
| 3bINP-100 | 0100651 | Hypoglycemia, type I diabetes mellitus | Diarrhea 4, malabsotive, congenital (OMIM #610370) | NEUROG3 | NM_020999.4:c.[117del];[117del] or p.[Thr40LeufsTer38];[Thr40LeufsTer38] | Autosomal dominant Wagner vitreoretinopathy (OMIM #143200) | VCAN | NM_004385.5:c.[3455C>A];[3455=] or p.[Ser1152Ter];[Ser=] | Incidental |
A Described genotypes considered only pathogenic and likely pathogenic variants. Novel variants are highlighted in bold.
Decisions taken in medical or nutritional management in the studied patients after WES by categories: (1) modification of the initial treatment, (2) continuation of the initial treatment, or (3) no treatment was provided before or after WES.
| Decision | Cause of Change or Mantainance | Patient ID | Initial Biochemical Diagnosis | Final WES Diagnosis | Initial Medical or Nutritional Management | Final Medical or Nutritional Management |
| (1) Modification of the initial treatment (n = 18) | Discordance between initial and final diagnosis | 3bINP-001 | Unspecific propionylcarnitine elevation; dysmorphological syndrome | Negative + Coffin-Siris syndrome type 10 (OMIM #618506) A✦ | B12 vitamin supplementation | Gradually B12 vitamin suspension as blood B12 levels normalized, plus closer monitoring by the orthopedics, cardiology, otorhinolaryngology, and neurology services. |
| 3bINP-041 | Hyper beta-alaninemia | Negative + Autosomal dominant lissencephaly type 1 (OMIM #607432) B✦ | B6 vitamin supplementation | Gradually B6 vitamin suspension, plus closer monitoring by neurology service. | ||
| A second disease found | 3bINP-045 | Hypoglycemia | AD Hyperinsulinemic hypoglycemia familial 1 + Autosomal dominant RET-related disorder (secondary finding) | Fasting avoidance | Continue with initial medical management, plus closer monitoring by oncology service, segregation analysis, and genetic counseling as the mother resulted heterozygous for RET pathogenic genotype | |
| 3bINP-047 | Unspecific acylcarnitine alterations | Alpha-methylacetoacetic aciduria + Autosomal dominant Cardiomyopathy, dilated, type 1G (secondary finding) | None | Initiation of nutritional treatment, plus referal to cardiology service for closer monitoring. | ||
| 3bINP-069 | MSUD | Maple syrup urine disease + ABCA4-related retinal distrophy (incidental finding) | Branched chain amino acids restricted diet | Continue with initial nutritional management, plus close monitoring by the ophthalmology service. | ||
| 3bINP-074 | 3-hydroxy-3-methylglutaric aciduria | 3-hydroxy-3-methylglutaric aciduria + Autosomal dominant MSH6-related Lynch syndrome (secondary finding) | Nutritional treatment, leucine and lipid restricted diet, carninite supplementation | Continue with initial nutritional management, plus closer monitoring by oncology service, segregation analysis, and genetic counseling as the father resulted heterozygous for MSH6 pathogenic genotype. | ||
| 3bINP-085 | Hypoglycemia | Hyperinsulinism hyperammonemia syndrome + X-linked glucose-6-phosphate dehydrogenase deficiency (expected finding) | Fasting avoidance. Diet high in complex carbohydrates such as corn starch, along with the recommended daily protein intake | Continue with initial nutritional management, plus diazoxide prescription, genetic counseling on risks of hemolytic anemia, and closer medical follow-up. | ||
| 3bINP-100 | Hypoglycemia, diabetes mellitus type 1 | Congenital diarrhea type 4 malabsorptive + Autosomal dominant Wagner vitreoretinopathy (incidental finding) | Fasting avoidance, insulin | Continue with initial medical management, plus close monitoring by the ophthalmology and gastroenterology services. | ||
| 3bINP-109 | Homocystinuria | Homocystinuria + Autosomal dominant Fleck corneal dystrophy (incidental finding) | Methionine restricted diet, betaine, B6 vitamin and folic acid supplementation, and monthly intake of B12 vitamin | Continue with initial nutritional management, plus close monitoring by the ophthalmology service. | ||
| Initial unspecific diagnosis + negative WES | 3bINP-006 | Suspicion of a FAOD for subtle elevation of tetradecanoylcarnitine and urinary excretion of adipic, suberic and sebacic acids | Negative | Long chain fatty acid restricted diet and medium-chain triglycerides supplementation | Gradual release from the nutritional management and redirection of the diagnostic approach | |
| 3bINP-016 | Suspicion of hypermethioninemia due to subtle elevation of blood methionine and homocysteine | Negative | Methionine restricted diet | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-024 | Suspicion of UCD because of hyperammonemia | Negative | Protein restricted diet, sodium benzoate and L-carnitine supplementaion | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-026 | Suspicion of hyperalaninemia because of elevation of blood alanine | Negative | Ketogenic diet | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-031 | Suspicion of hypermethioninemia because of 4-fold elevation of methionine and 3-fold elevation of Met/Phe ratio | Negative | Methionine restricted diet | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-050 | Suspicion of organic acidemia because of subtle propionylcarnitine elevation | Negative | B12 vitamin supplementation | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-056 | Suspicion of FAOD due to unspecific elevation of blood long chain acylcarnitines | Negative | Fasting avoidance and long chain fatty acid restricted diet | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-063 | Succinyl-CoA:3-oxoacid-CoA transferase deficiency due to elevated excretion of 3-hydroxybutiric and acetoacetic acids | Negative | Isoleucine restricted diet | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| 3bINP-072 | Suspicion of GSD due to positive liver biopsy | Negative | Fasting avoidance. Diet high in complex carbohydrates such as corn starch, along with the recommended daily protein intake | Gradual release from the nutritional management and redirection of the diagnostic approach | ||
| (2) Continuation of initial treatment (n = 5) | Monoallelic genotype found | 3bINP-036 | Organic acidemia for presence of 3-hydroxy-isovaleryl carnitine + methylmalonyl carnitine | Only one variant in HLCS gene | Biotin supplementation | Maintainance of biotin supplementation |
| 3bINP-052 | MCAD deficiency for the elevation of hexanoyl, octanoyl, and decanoyl carnitines | Only one variant in ACADM | Fasting avoidance | Maintainance of fasting avoidance | ||
| Genotype constituted of two VUS variants | 3bINP-076 | MSUD for remarkable blood elevation of branched chain amino acids and elevated excretion of branched chain keto acids in urine | Presence of two VUS variants in DBT | Branched chain amino acids restricted diet | Maintainance of branched chain amino acids restricted diet | |
| 3bINP-089 | Galactosemia for blood elevation of galactose, and normal activity of galactose-1P-uridyl transferase | Presence of two VUS variants in GALK1 | Galactose restricted diet | Maintainance of galactose restricted diet | ||
| 3bINP-094 | MSUD for remarkable blood elevation of branched chain amino acids and alloisoleucine, and elevated excretion of branched chain keto acids in urine | Presence of two VUS variants in DBT | Branched chain amino acids restricted diet | Maintainance of branched chain amino acids restricted diet | ||
| (3) No specific treatment was provided before or after WES (n = 9) | Not confirmated unspecific biochemical findings | 3bINP-007 | Suspicion of hyperprolinemia due to subtle elevation of blood proline | Negative | None | Redirection of the diagnostic approach |
| 3bINP-009 | Suspicion of a carbohydrate disorder due to hypoglycemia and hyperinsulinism | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-034 | Suspicion of FAOD due to subtly altered acylcarnitines profile | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-038 | Suspicion of MSUD because of subtle elevation of branched chain amino acids | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-039 | Suspicion of hyperprolinemia due to subtle elevation of blood proline | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-051 | Suspicion of FAOD due to unspecific altered acylcarnitines profile | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-070 | Suspicion of MSUD because of subtle elevation of branched chain amino acids | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-093 | Suspicion of MSUD vs organic acidemia for subtle elevation of branched chain amino acids and butyrylcarnitine | Negative | None | Redirection of the diagnostic approach | ||
| 3bINP-103 | Suspicion of organic acidemia for subtle elevation of 3-hydroxy-isovaleryl carnitine + hyperlactatemia | Negative | None | Redirection of the diagnostic approach |
A SOX4 genotype: NM_003107.3(SOX4):c.[1061C>A];[=] or p.[Ser354*];[=]. B PAFAH1B1 genotype: NG_009799.1(NM_000430.4):c.[116_117+2dup];[=] or p.[?];[=]. ✦ Syndromic entities not related to IEiM. Abbreviations: MSUD, maple syrup urine disease; FAOD, fatty acid oxidation disorder; GSD, glycogen storage disease; UCD, urea cycle disorder; MCAD, medium-chain acyl-CoA dehydrogenase deficiency.
Supplementary Materials
The following supporting information can be downloaded at:
References
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Abstract
Biochemical phenotyping has been the milestone for diagnosing and managing patients affected by inborn errors of intermediary metabolism (IEiM); however, identifying the genotype responsible for these monogenic disorders greatly contributes to achieving these goals. Herein, whole-exome sequencing (WES) was used to determine the genotypes of 95 unrelated Mexican pediatric patients suspected of having IEiM. They were classified into those bearing specific biochemical abnormalities (Group 1), and those presenting unspecific biochemical profiles (Group 2). The overall concordance between the initial biochemical diagnosis and final genotypic diagnoses was 72.6% (N = 69/95 patients), with the highest concordance achieved in Group 1 (91.3%, N = 63/69), whereas the concordance was limited in Group 2 (23.07%). This finding suggests that previous biochemical phenotyping correlated with the high WES diagnostic success. Concordance was high for urea cycle disorders (94.1%) and organic acid disorders (77.4%). The identified mutational spectrum comprised 83 IEiM-relevant variants (pathogenic, likely pathogenic, and variants of uncertain significance or VUS), including three novel ones, distributed among 29 different genes responsible for amino acid, organic acid, urea cycle, carbohydrate, and lipid disorders. Inconclusive WES results (7.3%, N = 7/95) relied on monoallelic pathogenic genotypes or those involving two VUS for autosomal-recessive IEiMs. A second monogenic disease was observed in 10.5% (N = 10/95) of the patients. According to the WES results, modifications in treatment had to be made in 33.6% (N = 32/95) of patients, mainly attributed to the presence of a second monogenic disease, or to an actionable trait. This study includes the largest cohort of Mexican patients to date with biochemically suspected IEiM who were genetically diagnosed through WES, underscoring its importance in medical management.
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Details
; Alcántara-Ortigoza, Miguel Angel 2
; González-del Angel, Ariadna 2
; Fernández-Hernández, Liliana 2
; Reyna-Fabián, Miriam Erandi 2
; Estandía-Ortega, Bernardette 2 ; Guillén-López, Sara 1
; López-Mejía, Lizbeth 1
; Belmont-Martínez, Leticia 1
; Carrillo-Nieto, Rosa Itzel 1
; Ibarra-González, Isabel 3
; Seung-Woo, Ryu 4
; Hane, Lee 4
; Fernández-Lainez, Cynthia 1
1 Laboratorio de Errores Innatos del Metabolismo y Tamiz, Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City C.P. 04530, Mexico
2 Laboratorio de Biología Molecular, Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City C.P. 04530, Mexico
3 Unidad de Genética de la Nutrición, Instituto de Investigaciones Biomédicas, UNAM, Mexico City C.P. 04530, Mexico
4 3billion, Inc., Seoul 03161, Republic of Korea