Metabolomics (2023) 19:49

https://doi.org/10.1007/s11306-023-02013-x

ORIGINAL ARTICLE

Application of machine learning tools and integrated OMICS for

screening and diagnosis of inborn errors of metabolism
Ganni Usha Rani1 · Srilatha Kadali1 · Banka Kurma Reddy1 · Dudekula Shaheena1 · Shaik Mohammad Naushad1

Received: 31 October 2022 / Accepted: 20 April 2023 / Published online: 3 May 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Introduction Tandem mass spectrometry (TMS) has emerged an important screening tool for various metabolic disorders in
newborns. However, there is inherent risk of false positive outcomes. Objective To establish analyte-specific cutoffs in TMS
by integrating metabolomics and genomics data to avoid false positivity and false negativity and improve its clinical utility.
Methods TMS was performed on 572 healthy and 3000 referred newborns. Urine organic acid analysis identified 23 types
of inborn errors in 99 referred newborns. Whole exome sequencing was performed in 30 positive cases. The impact of
physiological changes such as age, gender, and birthweight on various analytes was explored in healthy newborns. Machine
learning tools were used to integrate demographic data with metabolomics and genomics data to establish disease-specific
cut-offs; identify primary and secondary markers; build classification and regression trees (CART) for better differential
diagnosis; for pathway modeling.
Results This integration helped in differentiating B12 deficiency from methylmalonic acidemia (MMA) and propionic aci-
demia (Phi coefficient=0.93); differentiating transient tyrosinemia from tyrosinemia type 1 (Phi coefficient=1.00); getting
clues about the possible molecular defect in MMA to initiate appropriate intervention (Phi coefficient=1.00); to link pathoge-
nicity scores with metabolomics profile in tyrosinemia (r2=0.92). CART model helped in establishing differential diagnosis
of urea cycle disorders (Phi coefficient=1.00).
Conclusion Calibrated cut-offs of different analytes in TMS and machine learning-based establishment of disease-specific
thresholds of these markers through integrated OMICS have helped in improved differential diagnosis with significant reduc-
tion of the false positivity and false negativity rates.

Keywords Inborn errors of metabolism · Newborn screening · Tandem mass spectrometry · Machine learning ·
Integrated OMICS · Cut-off values

1 Introduction The spectrum of disorders that can be screened by NBS has

Newborn screening (NBS) is aimed at detecting most of
the Inborn errors of metabolism (IEM), including inher-
ited congenital endocrinopathies and metabolic disorders at
a very early stage. Enzyme or cofactor deficiencies cause
these diseases due to the accumulation of toxic metabolites,
which may cause irreversible organ damage in the first few
days of life. Early diagnosis and timely intervention prevent
the mortality and morbidity associated with these disorders.
Shaik Mohammad Naushad
naushadsm@gmail.com; naushad@yodalifeline.in
1
Department of Biochemical Genetics, YODA Lifeline
Diagnostics Pvt. Ltd, Ameerpet, Hyderabad 500016, India
increased tremendously after the introduction of tandem
mass spectrometry (TMS) [Harms et al. 2011], thus cover-
ing aminoacidopathies, fatty acid oxidation defects (FAOD),
carnitine cycle defects, and organic acidemias [Wilcken et
al. 2003]. The efficiency of NBS depends on the precision
in establishing analyte-specific thresholds that distinguish
physiological changes from pathological changes. This pre-
cision can be attained through continuous learning from the
data of confirmatory tests such as plasma amino acids and
urine organic acids [Dietzen et al., 2009]. Certain analytes
in TMS that are elevated in multiple metabolic disorders
where the differential diagnosis is possible only through
confirmatory testing or genomics.
Although there is no Government sponsored public
health program for mandatory NBS in India, a few studies

13
49 Page 2 of 10
have helped in identifying the high prevalence of treatable
metabolic disorders in India. The initial study was con-
ducted in the 1980s’ based on thin layer chromatography
(TLC) where 98,256 newborns were tested and 46 amino
acid disorders were identified with an incidence of 1 in
2136 for amino acidopathies (Rao et al., 1988). The first
systematic NBS was conducted in Andhra Pradesh, where
20,000 newborns born in all major government hospitals
of Hyderabad were tested for treatable IEMs. TLC and
High-Performance Liquid Chromatography (HPLC) were
used for amino acid analysis. This study showed an inci-
dence of 1:3660 for amino acidopathies (Rama Devi et al.
2004). TMS-based NBS has been popular in the last decade.
An initial study on 2550 clinically symptomatic children
showed a 3.2% positivity rate with 54% positive cases
showing amino acidopathies, 41.6% cases with organic aci-
demias, and 4.4% showing FAOD (Nagaraja et al., 2010). A
population-based study representing 4946 newborns from
the rural population of Andhra Pradesh identified 5 positive
cases, giving an incidence of 1:1000. The identified disor-
ders are Carnitine uptake disease, Isovaleric acidemia, Glu-
taric aciduria type-I, and Glutaric aciduria type-II (Sahai et
al., 2011). Although TMS is being performed in many cor-
porate hospitals currently, there are no comprehensive stud-
ies with inclusion of confirmatory and genetic tests for a
better genotype-phenotype correlation. In the current study,
we analyzed all screen-positive cases further using Gas
Chromatography-Mass Spectrometry (GC-MS) for organic
acid analysis. Whole exome sequencing (WES) was per-
formed to identify the causative genetic defects based on the
consent of the parents. Machine learning (ML) tools were
applied to correlate metabolomics data with genomics data.
Such correlation was explored for establishing differential
diagnosis criteria for certain metabolic disorders. It would
therefore be possible to initiate therapy without waiting for
the genetic report so that any damage to the organs can be
avoided.
In the current study, we have calibrated the cut-offs of
different analytes in TMS and used ML tools to provide
meaningful insights toward differential diagnosis through
integrated OMICS. It minimizes false positives and nega-
tives and guides toward a specific diagnosis.
2 Materials and methods
2.1 Study subjects
The baseline data were established on the basis of 572
newborns (0–7 months) with 234 females and 338 males
who were healthy and had no clinical symptoms sugges-
tive of metabolic disease during 3–4 months of follow-up.
13
G. Usha Rani et al.
The number of participants in each subgroup based on age
and gender were tabulated in Supplementary Table 1. The
mean birth weight was 2.72 ± 0.71 Kg. Subsequently, 3000
newborn samples referred from different hospitals were
screened for IEMs using TMS. Pre-analytical requirements
i.e., sample collection, storage, and transportation, and ana-
lytical requirements such as quality control, and reagent
stability were taken care of. Samples that did not pass pre-
analytical and analytical criteria were excluded from the
analysis. Whatman 903 grade filter paper was used for col-
lecting blood samples. The DBS samples were collected
from the heel prick of babies and were collected after 48 h
of birth following two feeds from the mother.
Urine samples were collected from patients with
informed consent from their parents. Frozen urine samples
or urine-soaked filter papers were recommended for sam-
ples received from the outstation.
2.2 LC-MS/MS analysis
The derivatized amino acid and acylcarnitine reagent kit
(ClinSpot® LC-MS/MS Complete Kit) was purchased from
Recipe (Part no. MS10000). This analytical method directly
determines 49 analytes of amino acids and acylcarnitines
(free carnitine, 35 acylcarnitines, and 13 amino acids), with-
out chromatographic separation on an HPLC column, via
tandem mass spectrometry (MS/MS) at a constant flow rate
of 0.1 ml/min. The runtime was 1 min with the isocratic
program. The detection was performed on an LC-MS/MS
system (LCMS-8045, Shimadzu Co., Ltd., Japan), which
was operated in positive ion mode (capillary voltage 2.0 kV,
desolvation temperature 526 °C). The internal standard is
used to calculate the analyte concentration of the sample.
Quantification of analytes was achieved using peak areas
that were processed using Neonatal software.
2.3 GC/MS analysis
Urine organic acid analysis was performed as per the pub-
lished protocols (Tanaka et al., 1980; Kimura et al., 1999).
Hydroxylamine hydrochloride, margaric acid, hydrochloric
acid, ethyl acetate, sodium sulphate, and sodium hydrox-
ide were purchased from Merck. Hydrocarbon mixture
(C10-26) was from GL Sciences and urease, N, O-bis
(trimethylsilyl)-fluoroacetamide (BSTFA) with 1% trimeth-
ylchlorosilane (TMCS), and tropic acid were procured from
Sigma Aldrich.
GC/MS system (Nexis GC-2030, GCMS-QP 2020 NX;
Shimadzu Co., Ltd., Japan) with the capillary column was a
fused silica Rtx-5MS (30 m X 0.25 mm) with 0.25 μm film
thickness of diphenyl dimethyl polysiloxane. Standard elec-
tron impact ionization scanning resulted in the Mass spectra
Application of machine learning tools and integrated OMICS for screening and diagnosis of inborn errors of…
in the range of m/z 35–600 at the rate of 0.4 s/cycle. The
temperature program was started at 100 °C with initial hold-
ing for 4 min and was increased at the rate of 4 °C/minute to
290 °C, with final holding for 10 min. The temperatures of
the injection port and interphase were both at 280 °C. The
flow rate of the helium carrier was 1.5 ml/min, and the linear
velocity was 40.2 m/sec. A final derivatized aliquot of 1 µl
was injected into GC/MS in spitless mode. Data acquisi-
tion was performed in the scan mode using a mass-to-charge
ratio. The run start time at 2.08 min and the end time was
60.86 min with the ion source temperature at 200 °C.
2.4 Whole exome sequencing
WES was performed on the next-generation sequencing
platform using the MGI machine (DNBSEQ-G50). The
Twist Bioscience Kit was used for preparing libraries for
WES. The concentration of each library was determined
using the QUBIT 2.0 Fluorometer. Samples were pooled
before sequencing with each sample at a final concentration
of 1.8 pM. Sequencing was performed on the DNBSEQ-
G50 platform using 150 cycles, paired-end chemistry by
targeting 100x coverage.
2.5 Machine learning algorithm: classification and
regression (CART) model
Demographic and metabolic data were used as the input
variables and the diagnosis was used as the output to iden-
tify disease-specific thresholds of acylcarnitines and amino
acids. The CART model was built as proposed by Leo Brei-
man. This is a binary tree algorithm with the most important
determinant at the apex of the tree with subsequent branches
formed with other variables in descending order of impor-
tance. Each root node was representative of a single input
variable. Branching was based on a specific threshold of
that variable that predict the outcome variable with reason-
ably. The extent of branching was optimized by pruning the
tree to achieve the required prediction with minimal branch-
ing. If multiple markers suggestive of a group of metabolic
disorders, differential diagnostic strategies were deduced
on the basis of these decision trees. The CART model was
validated using cases where molecular confirmation was
available.
2.6 Statistical analysis
The validity of each decision level of the CART model was
assessed on the basis of number of True Positives, True
Negatives, False Positives and False Negatives computed
in a 2 × 2 contingency table. Fisher exact test was performed
for the calculation of the performance characteristics of the
Page 3 of 10 49
model. We used student t-test for analyzing the impact of
gender-differences in each analyte. One-way ANOVA was
used to assess the changes in metabolites with growing age
(from birth to 6–7 months of life). Computational platforms
like https://statpages.info/ and https://www.socscistatistics.
com were employed for the analysis.
3 Results
3.1 Establishing population-specific reference
ranges for tandem mass spectrometry
The data from 572 healthy newborns were used to estab-
lish the population-specific reference ranges (1st percen-
tile − 99th percentile) of acylcarnitines and amino acids.
The upper limits of each analyte in controls were consid-
ered as the cut-off. Our cut-offs showed good correlation
(r2 = 0.906) with the cut-offs of the Center for Disease Con-
trol and Prevention (CDC).
Arginine, citrulline, and valine showed a positive corre-
lation with age. C2, C16, and C16:1 acylcarnitines showed
an inverse association with age while C8 and C4DC have a
positive association. No statistically significant gender dif-
ferences were observed in the amino acid and acylcarnitine
profiles. Except for C16 and C6DC, none of the analytes
showed statistically significant association with birth weight
after Bonferroni’s corrections (Table 1).
Gender differences in the distribution of glutamic acid,
glycine, C5, C6DC, C16OH, C14:1, and C16:1OH were
observed (Supplemental Table 2). Additionally, the refer-
ence ranges significantly changed from 1 month to 6 months
of age in infancy, specifically for the analyte’s aspartic acid,
citrulline, glutamic acid, glycine, phenylalanine, C16, C18,
C4OH, C4DC, C16:1, and C18:2 in females (Supplemen-
tal Table 3) similarly arginine, aspartic acid, glutamic acid,
glycine, leucine, ornithine, phenylalanine, C5DC, C16,
C18, C4DC, C16OH, C14:1, C16:1, C18:2, and C18:1 were
affected by age in males (Supplemental Table 4). These dif-
ferences can be attributed to altered metabolic rates with
age.
3.2 Incidence of IEMs among referred cases
The screen-positive cases on TMS were further confirmed
by urine organic acid analysis on GC/MS. The metabolite
elevations in the urine were consistent with the findings
observed on TMS. Among amino acid disorders, Tyrosin-
emia and Maple syrup urine disease are the most common,
followed by hypermethioninemia, urea cycle disorder, and
phenylketonuria (PKU). Among organic acidurias, Propi-
onic acidemia (PA) and Glutaric acidemia type-I (GA-1)
13
49 Page 4 of 10 G. Usha Rani et al.

Table 1 Indian-population spe- Analyte Min Max Correlation coefficient

cific cut-offs of amino acids and Age Male vs. female Birth weight
acylcarnitines and its correlation
with physiological variables Alanine 72.26 662.85 0.02 0.03 -0.05
Arginine 1.21 80.70 0.27* 0.07 -0.10
Aspartic acid 12.20 173.29 0.12 0.01 -0.10
Citrulline 4.12 60.53 0.28* -0.01 -0.09
Glutamic acid 113.30 594.93 -0.18* -0.03 0.02
Glycine 119.39 702.37 -0.12 -0.01 0.04
Leucine 50.50 388.27 -0.05 -0.04 0.09
Methionine 7.64 61.22 -0.01 -0.01 0.03
Ornithine 17.03 402.79 0.07 0.02 -0.10
Phenylalanine 20.28 118.65 0.05 -0.05 0.05
Proline 70.82 335.90 -0.04 0.01 0.02
Tyrosine 13.63 234.72 -0.08 -0.08 -0.02
Valine 36.10 318.57 0.16* 0.01 0.11
Free Carnitine (C0) 4.69 88.30 0.00 -0.01 -0.04
Acetyl Carnitine (C2) 3.61 96.95 -0.16* 0.05 0.08
Propionyl Carnitine (C3) 0.25 5.45 -0.03 0.03 0.12
Butyryl Carnitine (C4) 0.07 1.76 -0.01 -0.05 -0.03
Isovaleryl Carnitine (C5) 0.04 0.78 0.03 0.04 -0.07
Glutaryl Carnitine (C5DC) 0.01 0.46 -0.08 -0.07 0.04
Hexanoyl Carnitine (C6) 0.00 0.53 0.02 -0.03 -0.03
Octanoyl Carnitine (C8) 0.02 0.65 0.14* -0.03 0.00
Decanoyl Carnitine (C10) 0.02 0.50 -0.06 -0.01 -0.01
Dodecanoyl Carnitine (C12) 0.02 1.75 -0.05 0.04 -0.01
Tetradecanoyl Carnitine (C14) 0.03 0.75 -0.10 0.05 0.01
Palmitoyl Carnitine (C16) 0.35 7.5 -0.20* 0.01 0.14*
Stearoyl carnitine (C18) 0.15 3.13 -0.08 0.03 0.00
Malonyl Carnitine (C3DC) 0.01 0.25 -0.01 0.03 0.11
3-Hydroxybutyryl Carnitine (C4OH) 0.03 0.65 -0.09 0.01 0.05
Methylmalonyl Carnitine (C4DC) 0.06 0.84 0.25* -0.01 0.01
3-Hydroxyisovaleryl Carnitine (C5OH) 0.03 0.53 0.06 0.05 0.04
Methylglutarylcarnitine (C6DC) 0.00 0.11 -0.10 -0.04 0.14*
Octenoylcarnitine (C8:1) 0.01 0.37 0.04 0.04 -0.09
3-Hydroxypalmitoyl Carnitine (C16OH) 0.00 0.13 -0.07 0.03 0.00
3-Hydroxystearoyl carnitine (C18OH) 0.00 0.09 -0.04 0.00 -0.01
3-methylcrotonylcarnitine (C5:1) 0.01 0.26 -0.04 -0.02 0.04
Decanoyl Carnitine (C10:1) 0.01 0.26 0.07 -0.02 -0.03
Decadienoyl carnitine (C10:2) 0.00 0.08 -0.04 0.07 -0.05
Dodecenoylcarnitine (C12:1) 0.03 0.78 -0.10 -0.05 0.09
Tetradecadienoylcarnitine (C14:2) 0.00 0.62 0.06 0.03 -0.10
Tetradecenoyl Carnitine (C14:1) 0.01 0.86 -0.09 -0.01 0.04
3-OH-Tetradecenoylcarnitine (C14OH) 0.00 0.14 -0.10 0.01 0.02
Hexadecanoylcarnitine (C16:1) 0.03 0.94 -0.18* -0.03 0.10
3-Hydroxypalmitoleylcarnitine (C16:1OH) 0.01 0.23 0.03 0.02 -0.03
Octadecadienoylcarnitine (C18:2) 0.03 2.28 0.04 0.01 -0.11
3-Hydroxylinoleoylcarnitine (C18:2OH) 0.00 1.60 -0.02 -0.11 0.01
* p-values are statistically
3-OH-Octadecenoylcarnitine (C18:1OH) 0.01 0.15 -0.03 0.01 -0.04
significant after Bonferroni cor-
rection Linoleoyl Carnitine (C18:1) 0.29 3.29 -0.08 -0.01 -0.02

are the most common accompanied by Methylmalonic aci-
demia (MMA), beta-keto thiolase deficiency, Isovaleric
acidemia (IVA), Carnitine update disease, and 3-hydroxy-
3-methylglutaryl-CoA lyase deficiency (HMG-CoA lyase
deficiency). Among FAODs, MCAD is the most com-
mon followed by Carnitine palmitoyltransferase I (CPT I)
deficiency, Long-chain 3-hydroxyacyl-CoA dehydrogenase
(LCHAD) deficiency, and Short-chain 3-hydroxyacyl-CoA
dehydrogenase (SCHAD) deficiency. Among Urea cycle
disorders, Argininosuccinate synthetase 1 (ASSI) deficiency
is the most common preceded by Argininemia (Table 2).

13
Application of machine learning tools and integrated OMICS for screening and diagnosis of inborn errors of… Page 5 of 10 49

Table 2 Incidence rate of inborn errors of metabolism (IEMs) showed 87.5% accuracy in diagnosing B12 deficiency and
(n = 3000)
100% accuracy in segregating the data into normal, MMA,
Disorder No. of Inci-
confirmed dence or PA with overall precision of 98.68% (Fig. 1). In GA-1
cases and IVA patients, the cut-off value of C5DC and C5 were
Amino acid disorders > 0.6 µmol/L and > 2.18 µmol/L, respectively.
Phenylketonuria 2 1:1500 The most frequent amino acid abnormality in Indian new-
Maple syrup urine disease (MSUD) 5 1:600 borns is tyrosine elevation. We have established thresholds
Argininemia 4 1:750 of tyrosine and methionine that can differentiate transient
Citrullinemia 2 1:1500 Tyrosinemia from classical Tyrosinemia type I (Phi coef-
Hypermethioninemia 4 1:750
ficient: 1.00). Tyrosine > 235 µmol/L and methionine > 34
Tyrosinemia 7 1:430
µmol/L indicate Tyrosinemia type I (Fig. 2). Tyrosine lev-
Organic acid disorders
els between 167 and 234.72 µmol/L and methionine levels
Carnitine uptake defect (CUD) 2 1:1500
Propionic aciduria (PA) 8 1:360
between 17 and 61.2 µmol/L indicate transient tyrosinemia.
Methylmalonic aciduria (MMA) 6 1:500
Isovaleric acidemia (IVA) 2 1:1500 3.4 Differential diagnosis of Urea Cycle Disorders
Beta keto thiolase (BKT) 5 1:600 (UCDs)
Glutaric aciduria type-1 (GA-1) 8 1:360
3-hydroxy-3-methylglutaryl-CoA lyase (HMG- 2 1:1500 During our study, we identified 28 UCDs with molecular
CoA lyase) deficiency confirmation. Two cases with low levels of citrulline and
Fatty acid oxidation disorders arginine with orotic aciduria were diagnosed to have Orni-
Medium-chain acyl-CoA dehydrogenase 9 1:330
thine transcarbamylase deficiency (OTC c.533 C > T, OTC
(MCAD) deficiency
Carnitine palmitoyltransferase I (CPT I) 3 1:1000
c.275G > A). Two cases with normal urine organic acid
deficiency profile had NAGS (c.702-4delA) and CPS1 (c.446G > A)
Long-chain 3-hydroxyacyl-CoA dehydroge- 1 1:3000 deficiencies. The most frequent UCD is citrullinemia.
nase (LCHAD) deficiency Citrulline > 417.5 µmol/L indicated ASS1 deficiency
Short-chain acyl-CoA dehydrogenase (SCAD) 1 1:3000 (c.1088G > A, c.1168G > A). Citrulline < 417.5 µmol/L and
deficiency
glutamine can differentiate between ASS1 (n = 11) and ASL
Urea Cycle Disorders
(n = 4) where ASL showed glutamine > 376.95 µmol/L. In
N-acetylglutamate synthetase (NAGS) 1 1:3000
deficiency Argininemia (n = 9), the disease-specific threshold for Argi-
Carbamoyl phosphate synthetase I (CPS1) 1 1:3000 nine was > 195.7 µmol/L (ARG1 c.899 C > G).
deficiency
Ornithine transcarbamylase (OTC) deficiency 2 1:1500 3.5 Integration of OMICS in the differential
Argininosuccinate synthetase 1 (ASSI) 11 1:272 diagnosis of IEMs by ML
deficiency
Argininosuccinate lyase deficiency 4 1:750
Integration of demographic data with metabolomics and
Argininemia 9 1:330
genomics using ML has given subtype-specific thresh-
Total 99 1:30
olds for further differentiation of MMA cases. If the age
of onset is > 27 months, the most likely MMA subtype is
3.3 Utility of ML in the differential diagnosis of IEMs Cb1C deficiency. If the age of onset is < 9 months and the
C3/C2 ratio < 10.35, most likely to be MUT deficiency. C3/
C3 deficiency is the most common abnormality observed in C2 > 10.35 results in MUT or Cb1B deficiency. If the age
Indian children. Through ML, we have identified different of onset is between 9 and 27 months, MMA elevation < 221
disease-specific thresholds for C3 and C3/C2 ratio that helps folds suggests Cb1A deficiency. MMA elevation is > 221
in distinguishing normal children from those with vitamin folds and C3/C2 ratio in between 11.89 and 12.22 suggests
B12 deficiency and MMA or PA (Phi coefficient = 0.93). of Cb1A deficiency. In MUT deficiency, C3/C2 ratio > 12.33
C3 < 5.22 µmol/L and C3/C2 ratio in the range of 0.38–0.68 with significant elevation of MMA. This prediction model
suggests vitamin B12 deficiency. Patients with MMA have showed 100% specificity and sensitivity in the differential
a C3 > 5.22 ratio and a C3/C2 ratio between 0.68 and 3.2. diagnosis of MMA (Fig. 3). The sensitivity and specificity
In PA patients, the level of C3 is > 7.38 µmol/L and the C3/ of the standard procedure used in our laboratory were 95.8%
C2 ratio is 0.72–4.29. TMS analysis alone cannot differenti- and 98.7%, respectively. ML-based approach improved the
ate MMA and PA. However, urine organic acid analysis by sensitivity to 98.2% and 99.8%, respectively.
GC/MS will help in the differential diagnosis. These ML

13
49 Page 6 of 10
Fig. 1 Utility of C3 and C3/C2 ratio in the differential diagnosis of B12 deficiency, methylmalonic acidemia, and propionic acidemia
3.6 Pathway modelling through the integration of
genomics and metabolomics
We have used the SNAP2 scores of nine Tyrosinemia
type I cases showed the following FAH mutations namely
c.192G > T (n = 3), c.941 T > C (n = 1), c.983 A > G (n = 1),
c.998delA (n = 1), c.1159G > A (n = 1), c.1211G > A (n = 1)
and c.709 C > T (n = 1) as an index of pathogenicity. We
developed a multiple linear regression model with these
pathogenicity scores as output and various metabolites of
the tyrosine metabolic pathway, namely plasma tyrosine,
urinary succinyl acetone, urinary 4-hydroxy phenyl pyru-
vic acid (PHPPA), and urinary 4-hydroxy phenyl lactic
acid (PHPLA) as input variables. The deduced equation is
as follows:
FAH SNAP2 score = 2.6107+ (0.1362 × Tyrosine)
+ (0.0005 × Succinyl acetone)
+ (0.0426 × PHPLA) + (0.0133 × PHPPA)
This equation explained 91.94% variability in the pathoge-
nicity score based on the metabolomic profile (Fig. 4).
13
G. Usha Rani et al.
4 Discussion
The cut-offs established by the current study showed good
correlation (r2 = 0.846) with the cut-offs derived from rural
newborn data from India (Sahai et al., 2011). Similarly, the
cut-offs showed good correlation the CDC cut-offs also (r2:
90.6%). This is the first study from India demonstrating
physiological differences in amino acids and acylcarnitines
based on gender and age. Our results are consistent with
the findings of a Canadian study that reported significant
physiological changes in 50% of amino acids and 70% of
acylcarnitines during the neonatal period (Teodoro-Morri-
son et al., 2015). NBS in Southwest Colombia also reported
age-related differences in amino acids and acylcarnitines,
whereas no significant differences by gender were observed
(Cespedes et al. 2017). Our results are consistent with the
study by Hammarqvist et al., 2010 that demonstrated a posi-
tive association of branched-chain and basic amino acids
with increasing age, highlighting the requirement for these
essential amino acids during growth (Hammarqvist et al.,
2010). Furthermore, an association of acylcarnitine profile
with age is also corroborated with the findings of Cavedon
et al., 2005 in demonstrating a significant lowering of sev-
eral acylcarnitines with age. Similar to the findings of Ces-
pedes et al., 2017, no gender differences were observed in
Application of machine learning tools and integrated OMICS for screening and diagnosis of inborn errors of…
Fig. 2 Utility of tyrosine and methionine levels in distinguishing tran-
sient tyrosinemia from tyrosinemia type 1
Tyrosine (Tyr) and Methionine (Meth) levels were able to distinguish
transient tyrosinemia from tyrosinemia type 1. WES analysis revealed
the amino acid and acylcarnitine profile. The positivity rate
in referred cases in our study was 2.37%, while the positiv-
ity rate was 1.4% in another large-scale study (Babu et al.,
2015). This study is in agreement with our study in iden-
tifying methylmalonic aciduria, glutaric acidemia type 1,
propionic aciduria, maple syrup urine disease, phenylketon-
uria, and tyrosinemia as the most frequent IEMs (Hampe et
al., 2017). Only two analytes showed association with birth
weight i.e., C16 and C6DC. However, none of these mark-
ers are individually specific for any metabolic disorder. C16
elevation along with C18, C18:1 and C18:2 is an indicator
of CPT II deficiency or carnitine/acylcartinine translocase
deficiency. In view of both primary and secondary markers,
the diagnosis will not be affected by birth weight.
Classification and regression trees based on metabolite
levels and their mutual ratios have been used in newborn
screening for a long time. C8, C10, and C8/C2 were used for
detecting medium chain acyl CoA dehydrogenase (MCAD)
deficiency in a study from Belgium (Van den Bulcke et
Page 7 of 10 49
nine Tyrosinemia type I cases showed the following FAH mutations
namely c.192G > T, c.941T > C, c.983 A > G, c.998delA, c.1159G > A,
c.1211G > A and c.709 C > T. Three cases exhibited c.192G > T
mutation.
al., 2011). Phenylalanine hydroxylase deficiency, MCAD
deficiency, and methylcrotonyl CoA carboxylase (3-MCC)
deficiency were detected with > 95.2% and false-positive
rate < 0.001% with machine learning (Baumgartner et al.,
2005).
We have used ML tools to reduce the false positivity
rate in NBS by defining disease-specific thresholds, and by
identifying primary and secondary markers for differential
diagnosis. A recent study also used a similar approach with
a Random Forest machine learning classifier that minimized
the number of false positives for GA-1 by 89%, for MMA
by 45%, for OTCD by 98%, and for VLCAD deficiency by
2% (Peng et al., 2020). ML models based on diagnostic cut-
offs reduced the number of false positive cases from 21 to
2 for phenylketonuria, from 30 to 10 for hypermethionin-
emia, and from 209 to 46 for 3-MCC deficiency (Chen et
al., 2013).
The utility of integrating metabolomics data with genom-
ics has been investigated earlier by coupling untargeted
13
49 Page 8 of 10 G. Usha Rani et al.

Fig. 3 Integration of OMICs in

differential diagnosis of IEMs
with machine learning tools
Mutations in MUT, Cb1A, Cb1B,
and Cb1C have been identified
in established cases of MMA.
In all these cases, confirma-
tion data was available, which
formed the basis to develop a
classification and regression
model. Age of onset is the most
significant determinant with
Cb1C deficiency manifesting
after 27 months. MUT and Cb1B
mutations were early onset (< 9
months). If the age of onset is
between 9–27 months, MMA
levels and C3/C2 ratio can
differentiate MUT and Cb1A
deficiency. Cb1A deficiency is
associated with < 221 for MMA,
if elevation is > 221 C3/C2 ratio
will be between 11.89–12.33
in Cb1A where as in > 12.33 in
MUT. 1, 2, 3, and 4 represent
key decision thresholds. MUT:
p.A668P, p.R532H; p.A376Sfs*6,
p.R511*; Cb1A: p.Y24*,
c.562 + 1_562 + 2insT, Cb1B:
p.R191Q, p.D112*, p.R191W;
Cb1C: p.R132*

Fig. 4 Pathogenicity prediction

model for FAH gene mutations
using SNAP2 score as an index
of pathogenicity

metabolomics upstream or downstream to the primary reac-
tion with in silico-simulated WES results to increase the
diagnostic value (Kerkhofs et al., 2020). We have developed
a pathogenicity score prediction model using metabolomics
data in patients with tyrosinemia. In the current study, we
have integrated TMS, GC-MS, and genomics data to derive
a ML model capable of differential diagnosis. This model
could give possible clues about the subtypes of methylmalo-
nic acidemia based on the age of onset, C3/C2 ratio, and uri-
nary methylmalonic acid content. This information will be
of clinical utility in initiating the therapy as early as possible
as waiting for the molecular report may adversely affect the

13
Application of machine learning tools and integrated OMICS for screening and diagnosis of inborn errors of…
prognosis. A Korean study used a 259-gene targeted NGS
panel along with TMS to improve the diagnostic yield of
IEMs (Ko et al., 2018) and showed 100% concordance in
the results.
The major strengths of the current study are (i) integra-
tion of NBS, metabolomics, and genomics to improve dif-
ferential diagnostic potential; (ii) physiological changes
namely the influence of age, gender, and birth weight, etc.
on various analytes also evaluated. Integrated OMICS gave
more insights into differential markers both in screening
and confirmation, assisting in a specific diagnosis and early
intervention.
The incidence depicted in this study may not be repre-
sentative of population incidence as these subjects were
referred from various hospitals and were not part of a man-
datory NBS program. Hence, the sample size is limited
compared to population-based screening programs.
5 Conclusions
To conclude, this study provided baseline data for acylcarni-
tines and amino acids in Indian newborns and explored the
physiological changes attributed to age, gender, and birth
weight. Integration of screening with confirmatory pan-
els such as urine organic acid analysis and whole exome
sequencing helped in establishing unique genotype-meta-
bolic phenotype correlations, thus facilitating informed clin-
ical decisions. Compared to conventional NBS programs,
ML-based integration offers high specificity and sensitivity
in detecting these IEMs.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s11306-
023-02013-x.
Acknowledgements We would like to acknowledge all the families
who participated in the study. We thank all the clinicians who have
referred samples to YODA Lifeline Diagnostics Pvt. Ltd., Hyderabad.
We thank management for providing the necessary infrastructure for
the study.
Author Contribution URG and SK participated in the study design, in-
terpretation of data, statistical analysis, and drafting of the manuscript.
BKR and SD were involved in carrying out the experiments. SMN car-
ried out the conception and design of the study, interpretation of data,
statistical analysis, and final approval of the manuscript.
Declarations
Compliance with ethical standards All procedures performed in stud-
ies involving human participants were in accordance with the ethical
standards of the institutional and/or national research committee. This
was in accordance with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards.
Page 9 of 10 49
Informed consent Informed consent was obtained from each partici-
pant in the study.
Conflict of Interest The authors declare that they have no conflicts of
interest.
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