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Article

Concordance Between Biochemical and Molecular Diagnosis Obtained by WES in Mexican Patients with Inborn Errors of Intermediary Metabolism: Utility for Therapeutic Management

by
Marcela Vela-Amieva
1,†,
Miguel Angel Alcántara-Ortigoza
2,†,
Ariadna González-del Angel
2,
Liliana Fernández-Hernández
2,
Miriam Erandi Reyna-Fabián
2,
Bernardette Estandía-Ortega
2,
Sara Guillén-López
1,
Lizbeth López-Mejía
1,
Leticia Belmont-Martínez
1,
Rosa Itzel Carrillo-Nieto
1,
Isabel Ibarra-González
3,
Seung-Woo Ryu
4,
Hane Lee
4 and
Cynthia Fernández-Lainez
1,* on behalf of Rare Diseases Mexican Effort Group (RaDiMEG)
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
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(21), 11722; https://doi.org/10.3390/ijms252111722
Submission received: 12 August 2024 / Revised: 18 October 2024 / Accepted: 21 October 2024 / Published: 31 October 2024
(This article belongs to the Special Issue Exploring Rare Diseases: Genetic, Genomic and Metabolomic Advances)
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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.

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 (https://www.lovd.nl/, accessed on 21 June 2024). Three novel variants were found: NM_000159.4:c.1173_1174insT, or p.(Asn392Ter), in GCDH, NM_001370658.1:c.1352G>C, or p.(Cys451Ser), in BTD, and NM_005271.5:c.1466C>G, or p.(Pro489Arg), in GLUD1. The detailed information, classification by pathogenicity, and allele frequency are shown in Supplementary Table S1. Missense variants were the most common among organic acid (N = 18/29; 62%), urea cycle (N = 10/17; 58.8%), carbohydrate (N = 10/14; 71.4%), and lipid disorders (N = 2/4; 50%), whereas indel variants were the most common in amino acid disorders (7/19; 36.8%). Homozygosis was found in 32 of the 64 different identified genotypes (50%; Table 2).
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: https://www.ncbi.nlm.nih.gov/clinvar/?term=HLCS%5Bgene%5D&redir=gene, accessed on 20 May 2024). Moreover, our patient, 3bINP-036, received biotin at a dose of 20 mg per day, which improved his biochemical profile, which consisted of normalization of the hydroxy-pentanoylcarnitine (C5-OH) blood concentration and disappearance of the abnormal urinary organic acids (Table 3). However, since patient 3bINP-036 did not have the typical biochemical profile of holocarboxylase synthetase deficiency at diagnosis, which consists of elevated C5-OH, a urine organic acid profile with elevated lactic acid, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acid, and tiglylglycine [35], further studies are warranted to confirm or discard this disease.
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, https://www.lovd.nl/; accessed on 20 May 2024), dbSNP (https://www.ncbi.nlm.nih.gov/snp/; accessed on 20 May 2024), Genome Aggregation Database (https://gnomad.broadinstitute.org/; accessed on 20 May 2024), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/; accessed on 20 May 2024), and The Human Genome Mutation Database (https://www.hgmd.cf.ac.uk/; accessed on 20 May 2024), or to the best of our knowledge, in the literature: NM_000159.4(GCDH):c.1173_1174insT, or p.(Asn392Ter), NM_005271.5(GLUD1):c.1466C>G, or p.(Pro489Arg), and NM_001370658.1(BTD):c.1352G>C, or p.(Cys451Ser) (Table 2, Supplementary Table S1). The nonsense p.(Asn392Ter) variant in GCDH was considered pathogenic, whereas the missense variants in GLUD1 p.(Pro489Arg) and BTD p.(Cys451Ser) were considered LP.
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 (https://www.pediatria.gob.mx/; accessed on 27 October 2024). Demographic data were registered. Patients with methylmalonic/propionic acidemia or hyperphenylalaninemia were not included in this study, as they are included in other institutional protocols and/or their mutational spectrum has already been reported [23,55].
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, https://hpo.jax.org/; accessed on 20 February 2024) [62], such that the symptom similarity score could be calculated [63,64] between the patient’s phenotype and that expected for ~7000 rare genetic diseases. Only variants deemed clinically significant and relevant to the patient’s primary clinical indications at the time of variant interpretation were reported. The clinical geneticists manually evaluated all these variants, prioritized by the classification and symptom similarity score to select the most likely genotype responsible for the diagnosed or suspected monogenic disease.
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 (https://hgvs-nomenclature.org/stable/; accessed on 12 August 2024).

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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252111722/s1.

Author Contributions

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.

Funding

This research was funded by the Instituto Nacional de Pediatría, Secretaría de Salud (Recursos Fiscales 2022–2024, Programa E022 Investigación y Desarrollo Tecnológico en Salud, Ciudad de México, México). WES studies were funded by a 3Billion grant to M.V.-A.

Institutional Review Board Statement

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).

Informed Consent Statement

Written informed consent was obtained from the patients involved in the study or by their parents for the publication of this paper.

Data Availability Statement

Publicly available datasets were analyzed in this study. The data can be found here: ClinVar: https://www.ncbi.nlm.nih.gov/clinvar/, accessed on 20 May 2024; dbSNP: https://www.ncbi.nlm.nih.gov/snp/, accessed on 20 May 2024; Genome Aggregation Database (gnomAD) v.2.1.1: https://gnomad.broadinstitute.org/, accessed on 20 May 2024; Leiden Open Variation Database (LOVD) v.3.0: https://www.lovd.nl/, accessed on 21 June 2024; Online Mendelian Inheritance in Man (OMIM): https://www.omim.org/, accessed on 20 May 2024; The National Center for Biotechnology Information (NCBI): https://www.ncbi.nlm.nih.gov/gene, accessed on 20 May 2024, and The Human Gene Mutation Database (HGMD): https://www.hgmd.cf.ac.uk/, accessed on 20 May 2024). The data presented in this study are available upon reasonable request from the corresponding author. The clinical and molecular data of patients and their relatives are not publicly available due to restrictions to preserve their confidentiality, which was part of the signed informed consent of each patient. All the herein reported clinically relevant genetic variants along with the available deidentified phenotypic data were submitted to the publicly available database LOVD v.3.0, Leiden Open Variation Database (https://www.lovd.nl/, accessed on 21 June 2024).

Acknowledgments

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.

Conflicts of Interest

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.

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Ijms 25 11722 g001
Figure 1. Overall, WES diagnosis concordance of the entire study population and by study groups. Note that maximum concordance was reached for those patients bearing a well-defined biochemical phenotype (Group 1) for a specific IEiM (91.3%). Instead, only in 23.1% of patients bearing an unspecific biochemical alteration (Group 2) suggesting an underlying IEiM did WES achieve a diagnostic genotype of a specific IEiM. Abbreviations: IEiM, inborn errors of intermediary metabolism; WES, whole-exome sequencing; VUS, variant of uncertain significance; DBT, branched-chain acyl transferase E2 component; GALK1, galactokinase-1 deficiency; HLCS, holocarboxylase synthetase; ACADM, acyl-CoA dehydrogenase medium-chain.
Figure 1. Overall, WES diagnosis concordance of the entire study population and by study groups. Note that maximum concordance was reached for those patients bearing a well-defined biochemical phenotype (Group 1) for a specific IEiM (91.3%). Instead, only in 23.1% of patients bearing an unspecific biochemical alteration (Group 2) suggesting an underlying IEiM did WES achieve a diagnostic genotype of a specific IEiM. Abbreviations: IEiM, inborn errors of intermediary metabolism; WES, whole-exome sequencing; VUS, variant of uncertain significance; DBT, branched-chain acyl transferase E2 component; GALK1, galactokinase-1 deficiency; HLCS, holocarboxylase synthetase; ACADM, acyl-CoA dehydrogenase medium-chain.
Ijms 25 11722 g001
Ijms 25 11722 g002
Figure 2. Concordance between the initial biochemical diagnosis and WES results performed in 95 patients by type of disorder: (A) total, (B) Group 1, and (C) Group 2. Biochemically confirmed or suspected IEiM categories in the studied population by type of IEiM and by group before (light bars) and after WES (dark bars) in the overall study population (D), Group 1 (E), and Group 2 (F). Concordance+ between the initial and final diagnoses is the degree to which the initial biochemical diagnosis matches the final molecular diagnosis. Abbreviations: UCD, urea cycle disorders; OA, organic acid disorders; AA, amino acid disorders; CARB, carbohydrate disorders; LIPID, lipid defects. A p-value < 0.05 was considered to be statistically significant (* p < 0.05, ** p < 0.01, and **** p < 0.0001, ns: not statistically significant).
Figure 2. Concordance between the initial biochemical diagnosis and WES results performed in 95 patients by type of disorder: (A) total, (B) Group 1, and (C) Group 2. Biochemically confirmed or suspected IEiM categories in the studied population by type of IEiM and by group before (light bars) and after WES (dark bars) in the overall study population (D), Group 1 (E), and Group 2 (F). Concordance+ between the initial and final diagnoses is the degree to which the initial biochemical diagnosis matches the final molecular diagnosis. Abbreviations: UCD, urea cycle disorders; OA, organic acid disorders; AA, amino acid disorders; CARB, carbohydrate disorders; LIPID, lipid defects. A p-value < 0.05 was considered to be statistically significant (* p < 0.05, ** p < 0.01, and **** p < 0.0001, ns: not statistically significant).
Ijms 25 11722 g002
Ijms 25 11722 g003
Figure 3. WES-positive IEiM landscape found in the studied Mexican patients. Abbreviations:CoA: coenzyme A; Def: deficiency; HHH Sx: hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; MCAD: medium-chain acyl-CoA dehydrogenase deficiency, MSUD: maple syrup urine disease; OTC: ornithine transcarbamylase.
Figure 3. WES-positive IEiM landscape found in the studied Mexican patients. Abbreviations:CoA: coenzyme A; Def: deficiency; HHH Sx: hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; MCAD: medium-chain acyl-CoA dehydrogenase deficiency, MSUD: maple syrup urine disease; OTC: ornithine transcarbamylase.
Ijms 25 11722 g003
Table 1. Gene variants responsible for IEiM identified in Mexican patients, categorized by pathogenicity.
Table 1. Gene variants responsible for IEiM identified in Mexican patients, categorized by pathogenicity.
Type of DisorderDiseaseGeneTotal Number of VariantsPathogenicLikely PathogenicVariant of Uncertain Significance
Amino acid disordersMaple syrup urine disease (MSUD) type Ib (OMIM #620698)BCKDHB6420
MSUD, type II (OMIM #620699)DBT6312
MSUD, type Ia (OMIM #248600)BCKDHA2110
Homocystinuria, B6-responsive and nonresponsive types (OMIM #236200)CBS2200
Cystinosis, nephropathic (OMIM #219800)CTNS2020
Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (OMIM #238970)SLC25A151100
Organic acid disordersHMG-CoA lyase deficiency, 3-OH-3-methylglutaric acidemia (OMIM #246450) HMGCL7520
Glutaric Acidemia Type 1 (OMIM #231670)GCDH5500
Isovaleric acidemia (OMIM #243500)IVD5410
Biotinidase deficiency (OMIM #253260)BTD4310
Mitochondrial DNA depletion syndrome 9 (encephalomyopathic type with methylmalonic aciduria) (OMIM #245400)SUCLG12110
Beta-ketothiolase deficiency or mitochondrial acetoacetyl-CoA thiolase deficiency or alphamethylacetoacetic aciduria (OMIM #203750)ACAT13120
Holocarboxylase synthetase deficiency (OMIM #253270)HLCS1100
3-Methylcrotonyl-CoA carboxylase 2 deficiency (OMIM #210210)MCCC21010
Succinyl CoA:3-oxoacid CoA transferase deficiency (OMIM #245050)OXCT11100
Urea cycle disordersArgininemia (OMIM #207800)ARG18530
Citrullinemia (OMIM #215700)ASS16420
Ornithine transcarbamylase deficiency (OMIM #311250)OTC2200
Argininosuccinic aciduria (OMIM #207900)ASL1010
Carbohydrate disordersGlycogen storage disease Ia (OMIM #232200)G6PC15410
Glycogen storage disease IIIb (OMIM #232400)AGL1010
Glycogen storage disease Ib (OMIM: 232220)SLC37A42110
Diarrhea type 4, malabsorptive, congenital (OMIM #610370)NEUROG31100
Galactokinase deficiency with cataracts (OMIM #230200) GALK12002
Hyperinsulinemic hypoglycemia, familial, type 1 (OMIM #256450) ABCC8 1 1 0 0
Hyperinsulinism-hyperammonemia syndrome (OMIM #606762)GLUD11010
Hyperinsulinemic hypoglycemia, familial, type 2 (OMIM #601820)KCNJ111010
Lipid disordersAcyl-CoA dehydrogenase, medium chain deficiency (MCAD, OMIM #201450)ACADM3300
Hypercholesterolemia familial type 1 (OMIM #143890)LDLR1100
Total8354254
Table 2. Genotypic spectrum underlying IEiM in positive cases of the studied population.
Table 2. Genotypic spectrum underlying IEiM in positive cases of the studied population.
Amino Acid Disorders
Biochemical PhenotypeInitialy Suspected DiseaseResponsible Gene (Reference Sequence)Patient IDLOVD Individual Accession NumberGenotype AProtein ChangeFinal DiagnosisInheritance
Elevated circulating branched chain amino acid concentration (HP:0008344)MSUDBCKDHB (NM_183050.4)3bINP-066451632c.[152del];[152del]p.[Val51GlyfsTer21];[Val51GlyfsTer21]MSUD type Ib (OMIM #620698)AR
3bINP-080451644c.[564T>A];[564T>A]p.[Cys188Ter];[Cys188Ter]
3bINP-020451365c.564T>A(;)1087T>Ap.(Cys188Ter)(;)(Tyr363Asn)
3bINP-077451640c.853C>T(;)667G>Cp.(Arg285Ter)(;)(Gly223Arg)
3bINP-004450321c.[970C>T];[970C>T]p.[Arg324Ter];[Arg324Ter]
3bINP-013450471c.[1087T>A];[1087T>A]p.[Tyr363Asn];[Tyr363Asn]
DBT (NM_001918.5)3bINP-069451637c.[75_76del];[75_76del] Bp.[Cys26TrpfsTer2];[Cys26TrpfsTer2]MSUD type II (OMIM #620699)
3bINP-092451652
3bINP-104451662c.[263_265del];[263_265del]p.[Glu88del];[Glu88del]
3bINP-027451439c.[434-15_434-4del];[434-15_434-4del]p.[?];[?]
3bINP-081451645c.670G>T(;)434-15_434-4delp.(Glu224Ter)(;)(?)
BCKDHA (NM_000709.4)3bINP-062451630c.890G>A(;)1192G>Tp.(Arg297His)(;)(Glu398Ter)MSUD type Ia (OMIM #248600)
Hyperammonemia (HP:0001987)Gyrate atrophy or HHH SxSLC25A15 (NM_014252.4)3bINP-021451367c.[113_116dup];[113_116dup]p.[Phe40AspfsTer4];[Phe40AspfsTer4]Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (OMIM #238970)AR
Homocystinuria (HP:0002156)HomocystinuriaCBS
(NM_000071.3)		3bINP-046451595c.[572C>T];[572C>T] Cp.[Thr191Met];[Thr191Met]Homocystinuria (OMIM #236200)AR
3bINP-087451647
3bINP-109451663
3bINP-049451597c.[1126G>A];[1126G>A]p.[Asp376Asn];[Asp376Asn]
Corneal crystals (HP: 0000531), Fanconi syndrome (HP: 0011463) CCystinosisCTNS
(NM_004937.3)		3bINP-082451648c.[22_23del];[1036_1047del] Dp.[Ile8PhefsTer13];[Asp346_Phe349del]Cystinosis, nephropathic (OMIM #219800)AR
Organic acid disorders
Biochemical phenotypeInitialy suspected diseaseResponsible genePatient IDLOVD individual accession numberGenotype AProtein changeFinal diagnosisInheritance
Decreased circulating biotinidase concentration (HP:0410145)Biotinidase deficiencyBTD
(NM_001370658.1)		3bINP-054451617c.468G>T(;)1270G>Cp.(Lys156Asn)(;)(Asp424His)Biotinidase deficiency(OMIM #253260)AR
Biotinidase deficiency with atypical outcome, epilepsy3bINP-101451656c.[754T>G];[754T>G]p.[Trp252Gly];[Trp252Gly]
Biotinidase deficiency3bINP-053451616c.1270G>C(;)754T>Gp.(Asp424His)(;)(Trp252Gly)
Biotinidase deficiency3bINP-011450470c.[1270G>C];[1270G>C]p.[Asp424His];[Asp424His]
Elevated circulating acylcarnitine concentration (HP: 0045045), organic aciduria (HP:0001992)Multiple carboxylase deficiency3bINP-090451651c.[1352G>C];[1352G>C]p.[Cys451Ser];[Cys451Ser]
Organic aciduria (HP:0001992)Multiple carboxylase deficiencySUCLG1
(NM_003849.4)		3bINP-028451440c.40A>T(;)548T>Cp.(Met14Leu)(;)(Ile183Thr)Succinate-CoA ligase, alpha subunit deficiency (OMIM #245400)AR
Elevated circulating acylcarnitine concentration (HP:0045045)Multiple carboxylase deficiency3bINP-084451646
Increased circulating isovaleric acid concentration (HP:0033148)Isovaleric acidemiaIVD
(NM_002225.5,
NC_000015.9)		3bINP-057451600c.[149G>C];[149G>C]p.[Arg50Pro];[Arg50Pro]Isovaleric acidemia (OMIM #243500)AR
3bINP-019451363c.[850C>T];[850C>T]p.[Arg284Trp];[Arg284Trp]
3bINP-003450320c.[1065G>C];[1065G>C]p.[Lys355Asn];[Lys355Asn]
3bINP-079451643c.[1175G>A];[1175G>A]p.[Arg392His];[Arg392His]
3bINP-005450322g.[(?_40710329)_(40710462_?)del];[(?_40710329)_(40710462_?)del](homozygous exon 12 deletion)p.[?];[?]
3-methylglutaric aciduria (HP:0410051)3-hydroxy-3-methylglutaryl-CoA lyase deficiencyHMGCL
(NM_000191.3)		3bINP-042451460c.[109G>T];[109G>T]p.[Glu37Ter];[Glu37Ter]3-hydroxy-3-methylglutaryl-CoA lyase deficiency (OMIM #246450)AR
3bINP-035451457c.[112G>T];[112G>T]p.[Val38Phe];[Val38Phe]
3bINP-010450325c.121C>T(;)233C>Tp.(Arg41Ter)(;)(Ser78Phe)
3bINP-074451639c.230del(;)31C>Tp.(Val77GlyfsTer16)(;)(Arg11Ter)
3bINP-032451445c.[505_506del];[505_506del]p.[Ser169LeufsTer8];[Ser169LeufsTer8]
Concentration of glutaric acid in the urine above the upper limit of normal (HP:0003150)Glutaric aciduriaGCDH
(NM_000159.4)		3bINP-018451362c.263G>A(;)1204C>Tp.(Arg88His)(;)(Arg402Trp)Glutaric aciduria (OMIM #231670)AR
3bINP-023 451436c.[700C>T];[1173_1174insT] Gp.[Arg234Trp];[Asn392Ter]
3bINP-068451635c.[1082+31_1243+678del];[1082+31_1243+678del](homozygous whole exon 11 deletion)p.[Ala362Tyrfs*3];[Ala362TyrfsTer3]
Elevated circulating acylcarnitine concentration (HP:0045045)Organic aciduriaACAT1
(NM_000019.4)		3bINP-047451615c.[473A>G];[473A>G]p.[Asn158Ser];[Asn158Ser]Alpha-methylacetoacetic aciduria (OMIM #203750)AR
Organic aciduria (HP:0001992)Organic aciduria3bINP-017450485c.826+3_826+6del(;)200T>Gp.(?)(;)(Leu67Arg)
Organic aciduria (HP:0001992)3-Methylcrotonyl-CoA carboxylase deficiencyMCCC2
(NM_022132.5)		3bINP-059451602c.[1356G>A];[1356G>A]p.[Met452Ile];[Met452Ile]3-Methylcrotonyl-CoA carboxylase deficiency (OMIM #210210)AR
Ketosis (HP:0001946)Ketone bodies defectOXCT1
(NM_000436.4)		3bINP-060451603c.[1243del];[1243del]p.[Ile415TyrfsTer6];[Ile415TyrfsTer6]Succinyl-CoA:3-oxoacid- CoA transferase deficiency (OMIM #245050)AR
Urea cycle disorders
Biochemical phenotypeInitialy suspected diseaseResponsible genePatient IDLOVD individual accession numberGenotype AProtein changeFinal diagnosisInheritance
Hyperargininemia (HP:0500153)ArgininemiaARG1
(NM_000045.4)		3bINP-033451456c.3G>A(;)767_769delp.(Met1?)(;)(Glu256del)Argininemia (OMIM #207800)AR
3bINP-067451634c.61C>T(;)466-1G>C Ep.(Arg21Ter)(;)(?)
3bINP-078451642
3bINP-037451458c.61C>T(;)892G>Cp.(Arg21Ter)(;)(Ala298Pro)
3bINP-055451599c.[425G>A];[425G>A]p.[Gly142Glu];[Gly142Glu]
3bINP-073451638c.466-1G>C(;)787G>Tp.(?)(;)(Glu263Ter)
3bINP-105451658c.425G>A(;)871C>Tp.(Gly142Glu)(;)(Arg291Ter)
Elevated plasma citrulline (HP:0011966)CitrullinemiaASS1
(NM_054012.4)		3bINP-022451368c.[34A>G];[34A>G]p.[Ser12Gly];[Ser12Gly]Citrullinemia (OMIM #215700)AR
3bINP-106451659c.[256C>T];[256C>T]p.[Arg86Cys];[Arg86Cys]
3bINP-008450323c.256C>T(;)836G>A Fp.(Arg86Cys)(;)(Arg279GIn)
3bINP-065451631
3bINP-095451654c.256C>T(;)1194-19_1197dupp.(Arg86Cys)(;)(?)
3bINP-015450482c.970G>A(;)40G>Ap.Gly324Ser)(;)(Gly14Ser)
Orotic aciduria (HP:0003218)Ornithine transcarbamylase deficiencyOTC
(NM_000531.6)		3bINP-043451463c.[583G>A];[583=] (heterozygous female)p.[Gly195Arg];[Gly=] (heterozygous female)Ornithine transcarbamylase deficiency (OMIM #311250)X-linked
3bINP-048451596c.[803T>C];[0] (hemizygous male)p.[Met268Thr];[0] (hemizygous male)
Argininosuccinic aciduria (HP:0025630)Argininosuccinic aciduriaASL
(NM_000048.4)		3bINP-014450472c.[209T>C];[209T>C]p.[Val70Ala];[Val70Ala]Argininosuccinic aciduria (OMIM #207900)AR
Carbohydrate disorders
Biochemical phenotypeInitialy suspected diseaseResponsible genePatient IDLOVD individual accession numberGenotype AProtein changeFinal diagnosisInheritance
Abnormal hepatic glycogen storage (HP:0500030), hypoglycemia (HP:0001943)Glycogen storage disease type IG6PC1
(NM_000151.4)		3bINP-029451441c.379_380dup(;)1039C>Tp.(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-107451660c.[533C>T];[500G>A]p.[Pro178Leu];[Cys167Tyr]
Hepatomegaly (HP:0002240), hypoglycemia (HP:0001943)3bINP-102451657c.[809G>T];[809G>T]p.[Gly270Val];[Gly270Val]
Abnormal hepatic glycogen storage (HP:0500030), hypoglycemia (HP:0001943)Glycogen storage disease type IIIAGL
(NM_000642.3)		3bINP-025451437c.[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 IbSLC37A4
(NM_001164277.1)		3bINP-097451655c.82C>T(;)1130G>Ap.(Arg28Cys)(;)(Gly377Asp)Glycogen storage disease Ib (OMIM #232220)AR
Type 1 diabetes mellitus (HP:0100651)Mauriac syndrome, type 1 diabetes mellitusNEUROG3
(NM_020999.4)		3bINP-100451661c.[117del];[117del]p.[Thr40LeufsTer38];[Thr40LeufsTer38]Diarrhea type 4, malabsorptive, congenital (OMIM #610370)AR
Hypoglycemia (HP:0001943)HypoglycemiaABCC8
(NM_000352.6)		3bINP-045451461c.[2506C>T];[2506=] Hp.[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 deficiencyGLUD1
(NM_005271.5)		3bINP-085451650c.[1466C>G];[1466=]p.[Pro489Arg];[Pro=]Hyperinsulinism-hyperammonemia syndrome (OMIM #606762)AD
Hypoglycemia (HP:0001943)HypoglycemiaKCNJ11
(NM_000525.4)		3bINP-030451442c.[560C>T];[560=] Hp.[Ala187Val];[Ala=]Hyperinsulinemic hypoglycemia, familial, type 2 (OMIM #601820)AD
Lipid defects
Biochemical phenotypeInitialy suspected diseaseResponsible genePatient IDLOVD individual accession numberGenotype AProtein changeFinal diagnosisInheritance
Elevated circulating acylcarnitine concentration (HP:0045045)Acyl-CoA dehydrogenase deficiency (MCAD)ACADM
(NM_000016.6)		3bINP-044451464c.799G>A(;)959C>Ap.(Gly267Arg)(;)(Ser320Ter)MCAD (OMIM #201450)AR
Hypercholesterolemia (HP:0003124)Familial hypercholesterolemiaLDLR
(NM_000527.5)		3bINP-058451601c.[337dup];[337=]p.[Glu113GlyfsTer17];[Glu=]Familial hypercholesterolemia type 1 (OMIM #143890)AD
A Described genotypes considered only pathogenic and likely pathogenic variants (see Supplementary Table S1). B, E, F Two patients sharing the same indicated genotype. C Three patients sharing the same indicated genotype. D A second monogenic disease was found in this patient, showing rapid progressive renal failure despite early pharmacological treatment with cysteamine (see Table 4). G Allele c.1173_1174insT was initially not detected by WES due to low-coverage issues, but it was further identified by the whole-genome sequencing approach focused on the GCDH sequence. H An autosomal dominant mode of inheritance was supported by demonstrating an affected phenotype in the heterozygous father and a normal homozygous genotype in the healthy mother. Novel variants are highlighted in bold. Genotypes are presented by ascendent nucleotide numbers. LOVD: Leiden Open Variation Database v.3.0.
Table 3. Biochemical primary biomarkers related to unsolved cases (inconclusive and negative) in the studied population.
Table 3. Biochemical primary biomarkers related to unsolved cases (inconclusive and negative) in the studied population.
Study GroupPatient IDHPOBiomarkerConcentration (Reference Value)Suspected DiseaseGene (Reference Sequence, Encoded Protein)Variant 1 (Classification)Variant 2 (Classification)Conclusion
13bINP-03600450453-hydroxy-isovalerylcanitine + methylmalonylcarnitine1.36 μmol/L (0.83)Organic acidemiaHLCS (NM_001352514.2, Holocarboxylase synthetase)c.2361_2362insT or p.(Val788CysfsTer108) (pathogenic)Not identifiedInconclusive A
3bINP-0520045045Hexanoylcarnitine0.16 μmol/L (0.12)Medium chain acyl-CoA dehydrogenase deficiencyACADM (NM_000016.6, Medium chain acyl-CoA dehydrogenase)c.985A>G or p.(Lys329Glu) (pathogenic)Not identifiedInconclusive B
Octanoylcarnitine0.36 μmol/L (0.16)
Decanoylcarnitine0.37 μmol/L (0.21)
3bINP-0720500030GlycogenPositive liver biopsyGlycogen storage diseaseNot identifiedNot identifiedNot identifiedNegative
3bINP-0760008344Leucine + isoleucine1924 μmol/L (40–228)Maple syrup urine diseaseDBT (NM_001918.5, Dihydrolipoamide branched-chain transacylase) c.1210-3T>A or p.(?) (VUS)c.1210-3T>A or p.(?) (VUS)Inconclusive
Valine443 μmol/L (37–237)
AlloisoleucineNot determined
0001992Urinay organic acid profileElevated excretion of branched chain keto acids
3bINP-0890012024Galactose (with Galactose-1-P uridyltransferase normal activity)19.99 mg/dL (<12)GalactosemiaGALK1 (NM_000154.2, Galctose kinase)c.56C>A or p.(Ala19Asp) (VUS)c.182C>T or p.(Thr61Met) (VUS)Inconclusive
3bINP-0940008344Leucine + isoleucine3226 μmol/L (<253)Maple syrup urine diseaseDBT (NM_001918.5, Dihydrolipoamide branched-chain transacylase) c.1261G>T or p.(Gly421Trp) (VUS)c.1261G>T or p.(Gly421Trp) (VUS)Inconclusive
Valine1286 μmol/L (<282)
Alloisoleucine64 μmol/L (Not detectable)
0001992Urinay organic acid profileElevated excretion of branched chain keto acids
23bINP-0010004359Propionylcarnitine4.6 μmol/L (<2.5)Organic acidemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0060045045Tetradecanoylcarnitine0.33 μmol/L (<0.31)Organic acidemiaNot identifiedNot identifiedNot identifiedNegative
0001992Urinay organic acid profileElevated excretion of adipic, suberic and sebasic acids
3bINP-0070008358Proline393 μmol/L (<290)HyperprolinemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0090001943Glucose<40 mg/dL (70)Carbohydrate disorderNot identifiedNot identifiedNot identifiedNegative
0000842Hyperinsulinemia19.3 uU/mL (<2)
3bINP-0120001943Glucose<40 mg/dL (70)Carbohydrate disorderNot identifiedNot identifiedNot identifiedInconclusive C
0000842Hyperinsulinemia26.8 uU/mL (<2)
3bINP-0160003235Methionine99 μmol/L (9–42)HypermethioninemiaNot identifiedNot identifiedNot identifiedNegative
0002156Homocysteine8 μmol/L (0–6.4)
3bINP-0240001987Hyperammonemia117 μmol/L (9–35)Urea cycle disorderNot identifiedNot identifiedNot identifiedNegative
3bINP-0260003348Alanine1007 μmol/L (<605)HyperalaninemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0310003235Methionine209 μmol/L (<52), HypermethioninemiaNot identifiedNot identifiedNot identifiedNegative
Met/Phe ratioMet/Phe 4.2 (<1.4)
3bINP-0340045045Hexadecanoylcarnitine2.98 μmol/L (<2.4)Fatty acid oxidation defectNot identifiedNot identifiedNot identifiedNegative
Octadecenoylcarnitine2.45 μmol/L (<1.6)
3-hydroxy-octadecenoylcarnitine0.06 μmol/L (<0.03)
Octadecadienoylcarnitine0.69 μmol/L (<0.47)
3bINP-0380008344Leucine + isoleucine426 (<253)Maple syrup urine diseaseNot identifiedNot identifiedNot identifiedNegative
Valine446 μmol/L (<282)
Xleu (Leu + Ile)/Phe ratio7.12 (<3.95)
Xleu (Leu + Ile)/Ala ratio10.7 (<0.43)
Val/Phe ratio7.42 (<4.95)
3bINP-0390008358Proline338 μmol/L (<290)HyperprolinemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0410012556beta-Alanine8 μmol/L (<5)Hyperbeta-alaninemiaNot identifiedNot identifiedNot identifiedNegative
0020079beta-Alaninuria256 mmol/mol creatinine (<6)
0500138Serine 194 μmol/L (85-185)
0002154Glycine356 μmol/L (138-349)
3bINP-0500001992Propionylcarnitine10.7 μmol/L (<4.3)Organic acidemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0510045045Free carnitine134 μmol/L (<53)Fatty acid oxidation defectNot identifiedNot identifiedNot identifiedNegative
Propionylcarnitine29 μmol/L (<4.2)
Butyrylcarnitine1.5 μmol/L (<0.5)
Hexadecanoylcarnitine5.1 μmol/L (<2.2)
Tetradecanoylcarnitine0.41 μmol/L (<0.19)
Octadecanoylcarnitine2.7 μmol/L (<0.87)
Octadecenoylcarnitine6.5 μmol/L (<2.8)
3bINP-0560045045Free carnitine456 μmol/L (<87)Fatty acid oxidation defectNot identifiedNot identifiedNot identifiedNegative
Hexadecanoylcarnitine0.32 μmol/L (<0.23)
Octadecanoylcarnitine0.13 μmol/L (<0.1)
Free carnitine/(hexadecanoylcarnitine + octadecanoylcarnitine) ratio1013 (<69)
3bINP-0630001992Urinay organic acid profileElevated excretion of 3-hydroxybutiric and acetoacetic acidsOrganic acidemiaNot identifiedNot identifiedNot identifiedNegative
3bINP-0700008344Leucine + isoleucine265 μmol/L (<253)Maple syrup urine diseaseNot identifiedNot identifiedNot identifiedNegative
Valine303 μmol/L (<282)
0008358Proline439 μmol/L (<290)
3bINP-0930008344Leucine + isoleucine349 μmol/L (<253)Organic acidemiaNot identifiedNot identifiedNot identifiedNegative
Valine345 μmol/L(<282)
0045045Butyrylcarnitine0.52 μmol/L (<0.45)
3bINP-1030001992Urinay organic acid profileElevation of 2-hydroxybutiric and 3-OH butyric acid Organic acidemiaNot identifiedNot identifiedNot identifiedNegative
0001942Hyperlactatemia5.8 (1-3.3 mmol/L)
00450453-hydroxy-isovalerylcanitine + methylmalonylcarnitine1.11 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.
Table 4. Co-occurrence of two monogenic diseases due to expected, incidental, or secondary findings in the studied patients.
Table 4. Co-occurrence of two monogenic diseases due to expected, incidental, or secondary findings in the studied patients.
Study GroupPatient IDHPOObserved Biochemical Abnormality1st Disease DetectedGene Responsible of First DiseaseGenotype AIdentified Second Monogenic DiseaseGene Responsible of Second DiseaseGenotype AType of Finding
13bINP-02112026HyperornithinemiaHyperornithinemia-hyperammonemia-hyperhomocitrullinuria syndrome (OMIM #238970)SLC25A15NM_014252.4:c.[113_116dup];[113_116dup] or p.[Phe40AspfsTer4];[Phe40AspfsTer4]Autosomal dominant polydactyly, postaxial, types A1 and B (OMIM #174200)GLI3NM_000168.6:c.[3740_3743dup];[3740=] or p.[Cys1249AlafsTer3];[Cys=]Expected
0001987Hyperammonemia
3bINP-0540001992Biotinidase deficiencyBiotinidase deficiency (OMIM #253260)BTDNM_001370658.1:c.468G>T(;)1270G>C or p.(Lys156Asn)(;)(Asp424His)Autosomal dominant FGFR2-related disorder (OMIM *176943)FGFR2NM_000141.5:c.[923A>G];[923=] or p.[Tyr308Cys];[Tyr=]Expected
3bINP-0690008344Elevated circulating branched chain amino acid concentrationMSUD type II (OMIM #620699)DBTNM_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)ABCA4NM_000350.3:c.[2453G>A];[2453G>A] or p.[Gly818Glu];[Gly818Glu]Incidental
3bINP-07404100513-methylglutaric aciduriaHMG-CoA lyase deficiency (OMIM #246450)HMGCLNM_000191.3:c.230del(;)31C>T or p.(Val77GlyfsTer16)(;)(Arg11Ter)Autosomal dominant Lynch syndrome (OMIM #614350)MSH6NM_000179.3:c.[2150_2153del];[2150=] or p.[Val717AlafsTer18];[Val=]Secondary
3bINP-0820000531CystinosisNephropathic cystinosis (OMIM #219800)CTNSNM_004937.3:c.[22_23del];[1036_1047del] or p.[Ile8PhefsTer13];[Asp346_Phe349del]X-linked Alport syndrome type 1 (OMIM #301050)COL4A5NM_033380.3:c.[3088G>A];[3088=] or p.[Gly1030Ser];[Gly=]Expected
3bINP-1090002156HomocystinuriaHomocystinuria, B6-responsive and nonresponsive types (OMIM #236200)CBSNM_000071.3:c.[572C>T];[572C>T] or p.[Thr191Met];[Thr191Met]Autosomal dominant Fleck corneal dystrophy (OMIM #121850)PIKFYVENM_015040.4:c.[853_854del];[853=] or p.[Leu285PhefsTer19];[Leu=]Incidental
23bINP-0450001943HypoglycemiaAutosomal dominant form of Hyperinsulinemic hypoglycemia familial type 1 (OMIM #256450)ABCC8NM_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) RETNM_020975.6:c.[2410G>A];[2410=] or p.[Val804Met];[Val=]Secondary
3bINP-0470045045Inespecific acylcarnitine alterationsAlpha-methylacetoacetic aciduria (OMIM #203750)ACAT1NM_000019.4:c.[473A>G];[473A>G] or p.[Asn158Ser];[Asn158Ser]Autosomal dominant Cardiomyopathy, dilated, type 1G (OMIM #604145)TTNNM_001267550.2:c.[87470_87471del];[87470=] or p.[Leu29157GlnfsTer6];[Leu=]Secondary
3bINP-0850001943HypoglycemiaAutosomal dominant form of Hyperisulinism-hyperammonemia syndrome (OMIM #606762)GLUD1NM_005271.5:c.[1466C>G];[1466=] orp.[Pro489Arg];[Pro=] X-linked Glucose-6-phosphate dehydrogenase deficiency (OMIM #300908)G6PDHemizygous male for haplotype NM_001360016.2:c.[376A>G;202G>A];[0] or p.[Asn126Asp;Val68Met];[0]Expected
3bINP-1000100651Hypoglycemia, type I diabetes mellitusDiarrhea 4, malabsotive, congenital (OMIM #610370)NEUROG3NM_020999.4:c.[117del];[117del] or p.[Thr40LeufsTer38];[Thr40LeufsTer38]Autosomal dominant Wagner vitreoretinopathy (OMIM #143200)VCANNM_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.
Table 5. 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.
Table 5. 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.
DecisionCause of Change or MantainancePatient IDInitial Biochemical DiagnosisFinal WES DiagnosisInitial Medical or Nutritional ManagementFinal Medical or Nutritional Management
(1) Modification of the initial treatment (n = 18)Discordance between initial and final diagnosis3bINP-001Unspecific propionylcarnitine elevation; dysmorphological syndromeNegative + Coffin-Siris syndrome type 10 (OMIM #618506) A✦B12 vitamin supplementationGradually B12 vitamin suspension as blood B12 levels normalized, plus closer monitoring by the orthopedics, cardiology, otorhinolaryngology, and neurology services.
3bINP-041Hyper beta-alaninemiaNegative + Autosomal dominant lissencephaly type 1 (OMIM #607432) B✦B6 vitamin supplementationGradually B6 vitamin suspension, plus closer monitoring by neurology service.
A second disease found3bINP-045HypoglycemiaAD Hyperinsulinemic hypoglycemia familial 1 + Autosomal dominant RET-related disorder (secondary finding)Fasting avoidanceContinue 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-047Unspecific acylcarnitine alterationsAlpha-methylacetoacetic aciduria + Autosomal dominant Cardiomyopathy, dilated, type 1G (secondary finding)NoneInitiation of nutritional treatment, plus referal to cardiology service for closer monitoring.
3bINP-069MSUDMaple syrup urine disease + ABCA4-related retinal distrophy (incidental finding)Branched chain amino acids restricted dietContinue with initial nutritional management, plus close monitoring by the ophthalmology service.
3bINP-0743-hydroxy-3-methylglutaric aciduria3-hydroxy-3-methylglutaric aciduria + Autosomal dominant MSH6-related Lynch syndrome (secondary finding)Nutritional treatment, leucine and lipid restricted diet, carninite supplementationContinue 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-085HypoglycemiaHyperinsulinism 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 intakeContinue with initial nutritional management, plus diazoxide prescription, genetic counseling on risks of hemolytic anemia, and closer medical follow-up.
3bINP-100Hypoglycemia, diabetes mellitus type 1Congenital 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-109HomocystinuriaHomocystinuria + Autosomal dominant Fleck corneal dystrophy (incidental finding)Methionine restricted diet, betaine, B6 vitamin and folic acid supplementation, and monthly intake of B12 vitaminContinue with initial nutritional management, plus close monitoring by the ophthalmology service.
Initial unspecific diagnosis + negative WES3bINP-006Suspicion of a FAOD for subtle elevation of tetradecanoylcarnitine and urinary excretion of adipic, suberic and sebacic acidsNegativeLong chain fatty acid restricted diet and medium-chain triglycerides supplementation Gradual release from the nutritional management and redirection of the diagnostic approach
3bINP-016Suspicion of hypermethioninemia due to subtle elevation of blood methionine and homocysteineNegativeMethionine restricted dietGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-024Suspicion of UCD because of hyperammonemiaNegativeProtein restricted diet, sodium benzoate and L-carnitine supplementaionGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-026Suspicion of hyperalaninemia because of elevation of blood alanineNegativeKetogenic dietGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-031Suspicion of hypermethioninemia because of 4-fold elevation of methionine and 3-fold elevation of Met/Phe ratioNegativeMethionine restricted dietGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-050Suspicion of organic acidemia because of subtle propionylcarnitine elevationNegativeB12 vitamin supplementationGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-056Suspicion of FAOD due to unspecific elevation of blood long chain acylcarnitinesNegativeFasting avoidance and long chain fatty acid restricted dietGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-063Succinyl-CoA:3-oxoacid-CoA transferase deficiency due to elevated excretion of 3-hydroxybutiric and acetoacetic acidsNegativeIsoleucine restricted dietGradual release from the nutritional management and redirection of the diagnostic approach
3bINP-072Suspicion of GSD due to positive liver biopsyNegativeFasting avoidance. Diet high in complex carbohydrates such as corn starch, along with the recommended daily protein intakeGradual release from the nutritional management and redirection of the diagnostic approach
(2) Continuation of initial treatment (n = 5)Monoallelic genotype found3bINP-036Organic acidemia for presence of 3-hydroxy-isovaleryl carnitine + methylmalonyl carnitineOnly one variant in HLCS geneBiotin supplementationMaintainance of biotin supplementation
3bINP-052MCAD deficiency for the elevation of hexanoyl, octanoyl, and decanoyl carnitinesOnly one variant in ACADMFasting avoidanceMaintainance of fasting avoidance
Genotype constituted of two VUS variants3bINP-076MSUD for remarkable blood elevation of branched chain amino acids and elevated excretion of branched chain keto acids in urinePresence of two VUS variants in DBTBranched chain amino acids restricted dietMaintainance of branched chain amino acids restricted diet
3bINP-089Galactosemia for blood elevation of galactose, and normal activity of galactose-1P-uridyl transferasePresence of two VUS variants in GALK1Galactose restricted dietMaintainance of galactose restricted diet
3bINP-094MSUD for remarkable blood elevation of branched chain amino acids and alloisoleucine, and elevated excretion of branched chain keto acids in urinePresence of two VUS variants in DBTBranched chain amino acids restricted dietMaintainance of branched chain amino acids restricted diet
(3) No specific treatment was provided before or after WES (n = 9)Not confirmated unspecific biochemical findings3bINP-007Suspicion of hyperprolinemia due to subtle elevation of blood prolineNegativeNoneRedirection of the diagnostic approach
3bINP-009Suspicion of a carbohydrate disorder due to hypoglycemia and hyperinsulinismNegativeNoneRedirection of the diagnostic approach
3bINP-034Suspicion of FAOD due to subtly altered acylcarnitines profileNegativeNoneRedirection of the diagnostic approach
3bINP-038Suspicion of MSUD because of subtle elevation of branched chain amino acidsNegativeNoneRedirection of the diagnostic approach
3bINP-039Suspicion of hyperprolinemia due to subtle elevation of blood prolineNegativeNoneRedirection of the diagnostic approach
3bINP-051Suspicion of FAOD due to unspecific altered acylcarnitines profileNegativeNoneRedirection of the diagnostic approach
3bINP-070Suspicion of MSUD because of subtle elevation of branched chain amino acidsNegativeNoneRedirection of the diagnostic approach
3bINP-093Suspicion of MSUD vs organic acidemia for subtle elevation of branched chain amino acids and butyrylcarnitineNegativeNoneRedirection of the diagnostic approach
3bINP-103Suspicion of organic acidemia for subtle elevation of 3-hydroxy-isovaleryl carnitine + hyperlactatemiaNegativeNoneRedirection 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.
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Vela-Amieva, M.; Alcántara-Ortigoza, M.A.; González-del Angel, A.; Fernández-Hernández, L.; Reyna-Fabián, M.E.; Estandía-Ortega, B.; Guillén-López, S.; López-Mejía, L.; Belmont-Martínez, L.; Carrillo-Nieto, R.I.; et al. Concordance Between Biochemical and Molecular Diagnosis Obtained by WES in Mexican Patients with Inborn Errors of Intermediary Metabolism: Utility for Therapeutic Management. Int. J. Mol. Sci. 2024, 25, 11722. https://doi.org/10.3390/ijms252111722

AMA Style

Vela-Amieva M, Alcántara-Ortigoza MA, González-del Angel A, Fernández-Hernández L, Reyna-Fabián ME, Estandía-Ortega B, Guillén-López S, López-Mejía L, Belmont-Martínez L, Carrillo-Nieto RI, et al. Concordance Between Biochemical and Molecular Diagnosis Obtained by WES in Mexican Patients with Inborn Errors of Intermediary Metabolism: Utility for Therapeutic Management. International Journal of Molecular Sciences. 2024; 25(21):11722. https://doi.org/10.3390/ijms252111722

Chicago/Turabian Style

Vela-Amieva, Marcela, Miguel Angel Alcántara-Ortigoza, Ariadna González-del Angel, Liliana Fernández-Hernández, Miriam Erandi Reyna-Fabián, Bernardette Estandía-Ortega, Sara Guillén-López, Lizbeth López-Mejía, Leticia Belmont-Martínez, Rosa Itzel Carrillo-Nieto, and et al. 2024. "Concordance Between Biochemical and Molecular Diagnosis Obtained by WES in Mexican Patients with Inborn Errors of Intermediary Metabolism: Utility for Therapeutic Management" International Journal of Molecular Sciences 25, no. 21: 11722. https://doi.org/10.3390/ijms252111722

APA Style

Vela-Amieva, M., Alcántara-Ortigoza, M. A., González-del Angel, A., Fernández-Hernández, L., Reyna-Fabián, M. E., Estandía-Ortega, B., Guillén-López, S., López-Mejía, L., Belmont-Martínez, L., Carrillo-Nieto, R. I., Ibarra-González, I., Ryu, S. -W., Lee, H., & Fernández-Lainez, C., on behalf of Rare Diseases Mexican Effort Group (RaDiMEG). (2024). Concordance Between Biochemical and Molecular Diagnosis Obtained by WES in Mexican Patients with Inborn Errors of Intermediary Metabolism: Utility for Therapeutic Management. International Journal of Molecular Sciences, 25(21), 11722. https://doi.org/10.3390/ijms252111722

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