Research Article - Current Pediatric Research (2022) Volume 26, Issue 7
Spectrum of common and rare small molecule inborn errors of metabolism diagnosed in a tertiary care centre.
- *Corresponding Author:
- Suvarna Magar,Department of Pediatrics, MGM Medical College, Aurangabad, India, E-mail: firstname.lastname@example.org
Received: 04 July, 2022, Manuscript No. AAJCP-22-67546; Editor assigned: 05 July, 2022, PreQC No. AAJCP-22-67546(PQ); Reviewed: 11 July, 2022, QC No. AAJCP-22-67546; Revised: 18 July, 2022, Manuscript No. AAJCP-22-67546(R); Published: 29 July, 2022, DOI:10.35841/0971-9032.26.7.1481-1500.
Introduction: In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function, or to the effects of reduced ability to synthesize essential compounds.
Materials and Methods: Total 602 patients were screened in genetic clinic, out of which 112 patients were suspected with IEM. Here we have included data of 40 patients which were diagnosed as small molecule IEM based on TMS/GCMS gold standard and genetic testing in few of them.
Result: Out of 602 patients referred to genetic OPD, 40 patients were diagnosed with small molecule inborn errors of metabolism (6.6%). 112 patients underwent tandem mass spectrometry and urine gas chromatography mass spectrometry.
Discussion: We present the cases of IEM referred to genetic clinic from PICU, NICU, wards and OPD. Most common reason for referral was metabolic encephalopathy, followed by global developmental delay and seizure disorder with less common being hypoglycemia, hepatic failure etc.
Conclusion: By creating this data resource we aimed to leverage an overview of different common and rare IEMs found in our region, and document the genetic variants that are relevant to the diagnosis of IEMs.
Metabolism, Mass spectrometry, Gas chromatography, Hypoglycemia.
Inborn errors of metabolism form a large group of genetic diseases involving defects in genes coding for enzymes, receptors, cofactors etc. in metabolic pathways . In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function, or to the effects of reduced ability to synthesize essential compounds. Inborn errors of metabolism are now often referred to as congenital metabolic diseases or inherited metabolic disorders. Majority of the IEMs are inherited in an autosomal recessive manner.
While individually they are rare, collectively they are common with an overall incidence of greater than 1:1,000 . More than 350 different IEMs have been described to date, and most of these are rare diseases/conditions . IEMs generally lead to encephalopathy, developmental delays, disabilities and even death if left undiagnosed and untreated. It is well documented that extended newborn screening with use of tandem mass spectrometry will prevent irreversible neurological damage and infant mortality .
There has been a dramatic increase in understanding, novel diagnostic tests and treatment of these diseases in developed countries. Though Indians have also started utilizing Tandem Mass Spectrometry (TMS) as screening investigation for a suspected IEM or high risk newborn with symptoms, utility of this cost effective technique is still not widespread.
Using relatively simple tests involving the detection of amino acids and acylcarnitines in dried blood spots on filter paper, Tandem Mass Spectrometry (TMS) allows for rapid screening and diagnosis of more than 40 metabolic disorders in amino acids, organic acids, and fatty acid oxidation, substantially improving the efficiency and accuracy of early diagnosis [5,6]. Further confirmation by urine Gas Chromatography And Mass Spectrometry (GCMS) helps in immediate therapeutic intervention and prevention of further morbidity . Genetic testing has further helped to confirm the diagnosis and to opt prenatal testing in future pregnancies.
The present study is the comprehensive data analysis of tandem mass spectrometry and urine metabolic pattern for the diagnosis of IEM by GC/MS in samples received for high-risk IEM screening. Some are diagnosed based on genetic testing.
The current study also revealed that our region has common as well as very rare IEMs being prevalent. Also by creating this data resource we aimed to leverage an overview of different common and rare IEMs found in our region, and document the genetic variants that are relevant to the diagnosis of IEMs.
Materials and Methods
Total 602 patients were screened in genetic clinic, out of which 112 patients were suspected with IEM. Here we have included data of 40 patients which were diagnosed as small molecule IEM based on TMS/GCMS gold standard and genetic testing in few of them. This prospective descriptive study was conducted in MGM medical college in Aurangabad between October 2019 and September 2021. Children between Newborn to 18 years of age admitted in Ward, NICU and PICU with metabolic emergency and diagnosed to have IEM were included in the study.
We included children diagnosed for the first time as IEM during the hospital stay and those who were diagnosed earlier either during newborn period or in the genetic clinic. Diagnosis of IEM was based on biochemical, molecular analysis and/or MRI brain findings. Biochemical testing done to confirm IEM include Tandem Mass Spectrometry (TMS) for detecting abnormality in acylcarnitine profile and amino acid profile, and urinary Gas Chromatography-Mass Spectrometry (GC-MS) for detecting abnormality inorganic acids.
Molecular analysis was done by utilizing next generation sequencing based DNA testing and further validated by Sanger Sequencing. Other tests which were utilized for diagnosis are plasma amino acids, urine or CSF amino acids, urine pterin assay etc. Data were collected by history taking, examination and primary metabolic workup followed by biochemical and molecular testing. Clinical parameters collected include the age of presentation as a crisis, newly diagnosed or known entity, and age of initial diagnosis, sex, consanguinity, clinical signs, and symptoms during the presentation, biochemical, molecular and MRI brain findings, diagnosis, course in the ward in terms of death, or discharge.
Details on biochemical tests done and the molecular analysis carried on these patients were studied. We included molecular testing done either immediately after the diagnosis or later from stored DNA. We also got carrier status of parents done when pro-band sample was not available for molecular diagnosis. IEMs were categorized as protein, lipid or carbohydrate metabolic disorder, vitamin responsive disorders. Institutional Ethics Committee approval was obtained. Categorical data were expressed as number and percentage.
Out of 602 patients referred to genetic OPD, 40 patients were diagnosed with small molecule inborn errors of metabolism (6.6%). 112 patients underwent tandem mass spectrometry and urine gas chromatography mass spectrometry, out of which 32 patients were diagnosed with some IEM based on TMS, urine GCMS and/or genetic testing.
8 patients were diagnosed on the basis of MRI brain and/or Genetic testing. Total 40 patients were diagnosed with IEMs. 35 patients (87.5%) were below 2 years of age, and 5 patients (12.5%) were more than 2 years of age (Table 1).
|Demographic characteristics||No. of Patients(n)||Percentage (%)|
Table 1. Demographic characteristics.
28 patients (70%) were males, and 12 patients (30%) were females. Parental consanguinity was seen in 29 patients (72.5%) A positive family history or a previous death of a sibling was seen in 9 patients (22.5%). 21 (52.5%) patients presented with acute encephalopathy, which was followed by seizures as the presenting complaint in 19 (47.5%) patients. Global developmental delay and recurrent vomiting was seen in 9 patients each (22.5%), hypoglycemia in 5 patients (12.5%), hepatic failure in 3 patients (7.5%) (Table 2).
|Symptoms/signs||No.of Patients(n)||Percentage (%)|
|Global developmental delay||9||22.5|
Table 2. Common clinical presentations of IEM.
17 patients (42.5%) were found to have organic acidemias, 4 patients (10%) had fatty acid oxidation defects, 6 patients (15%) had disorder of amino acidopathies, 7 patients (17.5%) had mitochondrial diseases, 3 patients (7.5%) had urea cycle defects. Whereas, carbohydrate metabolism defects, purine metabolic defects and neurotransmitter metabolic defects had 1 patient in each group (2.5%) each (Table 3). The details of the disorders detected are shown in Table 4.
|Sr. No||Disease category||No.of patients diagnosed(n)||Percentage (%)|
|3||Fatty acid oxidation defects||4||10|
|4||Urea cycle defects||3||7.5|
|5||Carbohydrate metabolic defects||1||2.5|
|7||Neurotransmitter metabolic defects||1||2.5|
|8||Purine metabolic defects||1||2.5|
Table 3. Small molecule IEMs diagnosed.
|Case. No.||Age of symptom onset/sex||Presentation||Key biochemical findings(micromoles/lit) (normal values)||Diagnosis||Molecular diagnosis|
|1||9 months/female||GDD||TMS-Phenylalanine–1763, tyrosine–37.3, Ratio–20.5||Hyperphenylalanemia|
|GCMS-increased 2 hydroxy phenyl lactic acid, 3 phenyl lactic acid|
|2||3 years 3 months/male||Distension of abdomen, recurrent infections, hepatosplenomegaly, bicytopenia||GCMS-lysine 3-3456(NMT 998)||Lysin uric protein intolerance||Homozygous likely pathogenic variant in SLC7A7 gene in exon 3 c.110 dupT|
|Lysine 4–2976(NMT 2646)|
|3||1 year 3 months/male||Failure to thrive, loose stools, hepatosplenomegaly, vitamin d deficiency, acute liver failure||TMS-proline–314(33-301), methionine–75.6(4.77-46), tyrosine–415(18.9-152), Ratio- 0.23,||Tyrosinemia type I||Homozygous likely pathogenic variant in FAH gene in exon 2 c.192 G>T tyrosinemia type I|
|GCMS- glutaric-2 12.21(8.44), succinyl acetone-OX-1–13.13().5), phenyllactic-2–12.45(5.79), 4-OH phenylacetic-2–791(12.51)|
|4||6 months/male||GDD, hypotonia||TMS- phenylalanine–1896(25-105), phenylalanine/tyrosine ratio–19.07(0.29-4.31)||Hyperphenylalanemia||Homozygous pathogenic variant, in exon 4, c.200C>T (pThr67Met) in PTS gene, causing Hyperphenylalaninemia, BH4-deficient, A|
|GCMS- phenyl pyruvic OX 2 -236.5(0), phenylacetic 1 -3.17(0-0.4), thiodiglycolic – 1.62(0), phenyl lactic 2 – 427(0-4.9), 4OH phenllactic2 – 194.15(0-7)|
|Plasma amino acid- glutamine- 170(246-1182)|
|5||3 months/female||GDD, lethargy, metabolic acidosis, ketonuria||TMS- methionine–3.6(4.6-48), acetyl carnitine–2.32(2.49-62.79), butyryl carnitine–0.04(0.06-1.3), propionyl carnitine/acetyl carnitine–0.64(0-0.5)||Hyperhomocystenemia|
|GCMS–methyl citric acid-4–2183 (NMT2014), methyl citric acid-4–24.85(NMT24), methylmalonic acid-2–8594(NMT1216)|
|6||15 days/male||Convulsions, encephalopathy, acute liver failure||GCMS- succinyl acetone–163.9(20-100)||Tyrosinemia I|
|7||7 months/female||Breathlessness and drowsiness, encephalopathy, ketonuria||TMS- tiglycarnitine(C5:1)-0.24 (>0.14)||2 methyl 3 hydroxy butyric aciduria|
|Malonyl carnitine/Hydroxy butyryl carnitine-C3DC/C4OH-1.98(>0.5)|
|Urine GCMS- 2 methyl 2 hydroxy butyric acid-2>569.3|
|3 OH isovaleric acid-2-853.19 (<330)|
|Hydroxybutyric acid 2|
|8||1 year 5 months/female||Loose stools, breathlessness, lethargy, unconsciousness, encephalopathy||GCMS-2-hydroxy glutaric acid 3–505(NMT363.42), glutaric acid–82.33(NMT 65.13),||2- hydroxy Glutaric aciduria|
|9||1 year 4 months/male||Fever, breathlessness, unconsciousness, encephalopathy||TMS- leucine/isoleucine/hydroxyproline–677(23.93-383), valine–616(31.28-450), free carnitine-6.38(7-109), propionylcarnitine-12.2(0.12-6.65), tiglycarnitine-0.15(0-0.14), glutamic acid-34.4(47-441), acetylcarnitine-0.73(0-0.55)||Propionic aciduria|
|GCMS-2- methyl butyryl glycine- 42.41(NMT 39.53), 3 hydroxy propionic acid-2–1163(NMT 473), 3 methyl glutaconic acid(E) 2 -363(NMT 271), 3 methyl glutaconic acid(Z) 2–253(NMT 101), glutaric acid 2–820(NMT116), methyl citric acid-4-7345(NMT2014),|
|10||2 months/male||Unconsciousness, encephalopathy||GCMS- 5-oxoproline-2–26059(NMT 704)||Oxoprolinuria|
|11||4 month /male||Metabolic encephalopathy||TMS- increased C8, alanine, decreased free carnitine||Glutaric aciduria II|
|12||10 months/female||Fever, vomiting, lethargy, encephalopathy||TMS- propionylcarnitine5.43(0.12-6.65)||Methylmalonic acidaemia|
|GCMS- 3 methyl glutaconic acid (z)-2–108.78(NMT101.54), methylmalonic acid-2–9612.58(NMT1216)|
|13||10 years/female||Altered sensorium, decreased oral intake, metabolic encephalopathy||TMS- acyl carnitines–7.61(9-51), total carnitines–14.81(31-123)||Glutaric aciduria II|
|GCMS- more than 4-fold increase in 3 hydroxybutyric acid, ethylmalonic acid, glutaric acid, fumaric acid, malic acid, acetyl glycine, tiglyglycine, glutaconic acid, tyrosine metabolite, 4 hydroxy phenyl lactic acid|
|14||13 months/male||Convulsions encephalopathy,||GCMS- 2 OH glutaric 3 – 132(0.6-5.9)||Glutaric aciduria type II|
|15||5 months/male||Convulsion, altered sensorium, encephalopathy, metabolic acidosis||TMS- glutaryl carnitine – 1.01(0-0.35)||Glutaric aciduria type I|
|16||15 days/male||Altered sensorium, encephalopathy||TMS- increased lactate, decreased carnitine level||Propionic aciduria|
|GCMS- increased glutaric acid, ethylmalonic acid|
|17||2 year 3 months/male||GDD||GCMS- lactate – 21.3(2-12), pyruvate – 0.96(0.2-2), increased 2 OH butyric acid, 3 OH butyric acid -2, 2 keto 3 methyl valeric acid -2 , adipic 2(C6), C8, 4 OH phenyl lactic, 4 OH phenyl pyruvate||3 methyl gluta conic aciduria type 5||EXOME-homozygous likely pathogenic variant in exon 5 of DNAJC19 gene causative of 3 methylglutaconic acidura type 5|
|18||2 years/male||Convulsions, altered sensorium, encephalopathy||TMS-negative||Biotin responsive basal ganglia disease||Exome- likely pathogenic homozygous variant in SLC19A3 gene in exon 3, c.595 T>A causing biotin responsive basal ganglia disease|
|Urine GCMS- negative|
|19||21 days/male||Breathlessness, excessive cry, convulsions, encephalopathy||TMS- increased methyl malonyl carnitine, propionyl carnitine||HMG coA lyase deficiency|
|GCMS- increased 3 OH methylglutaconic acid, 3 OH isovaleric acid,|
|20||9 days/female||Refusal to feed and decreased activity||TMS – increased leucine and valine||Maple syrup urine Disease||Exome- both parents’ carrier of heterozygous likely pathogenic variant in BCKDHA gene in exon 9 c.1251delC|
|21||10 days/male||Encephalopathy, metabolic acidosis, ketonuria||TMS-C3-C3/C0 ratio||Propionic acidaemia|
|Urine GCMS-increased 3 hydroxy propionate, methyl citrate, and 3 hydroxy isovalerate and significant ketonuria|
|22||3 days/male||Encephalopathy||TMS - C3 - 11.19, C3/C2 RATIO - 0.63, C3/C6 - 7.1||Methylmalonic aciduria||Heterozygous likely pathogenic|
|Variant 1 – MMUT gene on exon 3, c.643G>T, p. Gly215Cys,|
|Variant 2 – MMUT gene on exon 3, c.692dup, p. Tyr231Ter|
|23||16 months/male||GDD||TMS-leucine/isoleucine/hydroxyproline-3190(23.9-383.0), valine - 937(31-450), malonylcarnitine-1.15(0-1)||MSUD||EXOME-Homozygous likely pathologic variant in exon 7 c.868 (p. Gly290Arg) in BCKDHA gene causative of MSUD|
|Urea cycle defects|
|24||8 days/male||Convulsion , metabolic encephalopathy||TMS–increased malonyl carnitine, citrulline–2250(93), citrulline/arginine ratio– 477.1(4), increased glutamine and methionine||Citrullinemia type I|
|25||3 years 10 months/ male||Decreased activity, lethargic||TMS - citrulline – 1180 (93), citrulline/arginine - >10% (4%)||Citrullinemia type I|
|26||8 days/female||Encephalopathy||TMS – citrulline – 2230(93), glutamine – 3240(1334), methionine – 177(65)||Citrullinemia type I||EXOM – homozygous likely pathogenic variant in ASS1 gene in exon 15, c.1168G>A causative of classic citrullinemia type I|
|Fatty acid oxidation defects|
|27||3 months/male||Distension of abdomen, vomiting, difficulty in breathing||TMS- alanine 78.2(93-1230), methionine 3.83(4.6-48), ornithine-21(25.14-330), argino succinic acid-3.77(0-2), glutamic acid-49.6(69.76-652), malonylcarnitine-2.25(0-0.5)||3 OH acyl CO A dehydrogenase deficiency|
|GCMS-3-hydroxyadipic acid-3 -1332(NMT 597), glycerol 3 phosphate-4 – 608(NMT203)|
|28||6 months/female||Motor developmental delay, hypoglycemia, deranged LFT||TMS- 0.62(0.01-0.34) C6 and C8-4.02(0.01-.038)||Medium chain Acyl CoA Dehydrogenase deficiency|
|GCMS-C6-440% (<30%) non hydroxy dicarboxylic acid C8-29.49% (<0.59%), C10-450% (<11.5%), C12-4.22% (<0.5%)|
|29||5 months/male||Global developmental delay, seizures, encephalopathy, metabolic acidosis, hyperlactatemia||C16 OH-0.19 (0.00-0.08)||Long chain 3Hydroxy acyl Co A dehydrogenase Deficiency|
|C18OH- 0.84 (0.07-0.15)|
|UOA-short chain, medium chain fatty acids and long chain fatty acids only up to C12 (dodecanedioic acid) and their corresponding 3 hydroxy dicarboxylic acids.|
|30||2 years/male||Recurrent vomiting and Ketotic hypoglycemia||TMS- C16OH- 0.19 (0.00-0.08)||Long chain acyl Co A dehydrogenase deficiency|
|UOA-2-Hydroxy-butyric acid and 3-Hydroxy-butyric acid|
|Adipic acid and 3-Hydroxy-dodecanedioic acid|
|Normal growth hormone, insulin, and cortisol levels|
|31||2 months/male||Yellowish discoloration of body, distension of abdomen, decreased activity, hypoglycemia||TMS- free carnitine – 1.34(7-121), acetyl carnitine – 2.08(2.49-62), butyryl carnitine- 0.05(0.06-1.3)||Carnitine uptake defect|
|32||9 months/Male||Loose stools, difficulty in breathing, hypoglycemia||TMS- free carnitine – 4.06(7-121), acetyl carnitine – 1.3(2.49-62.79), propionyl carnitine – 0.1(0.12-6.65), butyryl carnitine – 0.05(0.06-1.3)||Carnitine uptake defect|
|33||7 years/female||Vomiting, loose stools, repeated episode of hypoglycemia||Decreased free carnitine, increased 3 FFA, increased 3 hydroxybutyrate||Carnitine uptake defect|
|34||2 years/male||GDD, convulsions, encephalopathy||TMS- negative||Mitochondria disease||MRI brain – T2 weighted hyperintensities in B/L central tegmental tracts in dorsal pons s/o mitochondrial disease|
|35||2 years/female||Motor developmental delay, vision loss, fundus shows optic atrophy||TMS-negative||Mitochondria DNA depletion syndrome – 7||EXOM-compound heterozygous variant TWNK gene|
|One heterozygous likely pathogenic variant in exon 1, c.1003 C>A and a second VUS in exon 5- c.2050 A>C causing mitochondrial DNA depletion syndrome 7|
|36||3 years/male||Convulsions, altered sensorium, encephalopathy, breathlessness||TMS-negative||Mitochondrial encephalopathy with lactic acidosis||MRI – T2 weight images shows B/L hyperintensities in bilateral, basal, ganglia, midbrain, pons and cerebellum, lactate peak on MRS mitochondrial encephalopathy|
|37||18 months/male||Convulsions, altered sensorium, encephalopathy, neuro regression||Sr. lactate – 2.6||Leigh disease||MRI brain – symmetrical T2 weighted hyperintensities in bilateral, basal, ganglia, midbrain, pons and cerebellum, lactate peak on MRS|
|Neurotransmitter metabolic defects|
|38||5 months/female||GDD with hypotonia||GCMS-2 deoxy tetronic acid-3 – 3688.36(NMT 806), 4 hydroxy butyric acid-2 – 11959(NMT 442), adipic acid-2 – 401(NMT 296), glutaric acid-2 – 589(NMT116), glycolic acid-2 – 1348(NMT 1238)||Succinic semialdehyde dehydrogenase deficiency||EXOME – homozygous likely pathogenic variant in ALDH 5A1 exome 4 c.701 C>T succinic semialdehyde dehydrogenase deficiency|
|Disorder of purine metabolic defect|
|39||17 months/male||GDD, convulsions, encephalopathy, microcephaly, hypotonia||TMS- negative||Inosine triphosphate phosphohydrolase deficiency||EXOM- ITPA gene detected in homozygous likely pathogenic variant in exon 3 c.137delA (p. Gln46Argfs Ter43) causative of inosine triphosphate phosphohydrolase deficiency (pathogenic variant) AR|
|Urine GCMS- negative|
|MRI- diffusion restriction in posterior limb of internal capsule|
|Disorders of carbohydrate metabolic defect|
|40||4 months/female||Yellowish discoloration of body, distension of abdomen, clay color stools||TMS-arginine – 67(0.78-60)||Classical galactosemia||compound heterozygous variant in GALT gene variant 1- likely pathogenic c.142 C>T in exon 2,|
|GCMS-galactitol-6- 1827(NMT 788), glycerol 3 phosphate-310(NMT 203), xanthine – 1412(NMT 323)||Variant 2- pathogenic c.610 C>T in exon 7 causative of classical galactosemia|
|Time resolved flouro immunoassay – T GAL – 30(NMT 25)|
Table 4. Disease spectrum of different types of Inborn Errors of metabolism.
We present the cases of IEM referred to genetic clinic from PICU, NICU, wards and OPD. Most common reason for referral was metabolic encephalopathy, followed by global developmental delay and seizure disorder with less common being hypoglycemia, hepatic failure etc. IEM could potentially be under-diagnosed and high index of suspicion and team effort is essential to diagnose IEM. Availability of advanced biochemical testing helped in the definitive diagnosis. The article on testing modalities of IEMs has really helped us to reach the diagnoses . Metabolite pattern recognition in all the tests helps to arrive at a specific diagnosis. TMS, GCMS, and HPLC of amino acids in blood and urine are the most common diagnostic modality to aid in definitive diagnosis [9,10].
The most common IEM group in our study was organic acidemias, accounting for 42% of the total IEM. Aminoacidopathies, organic acidemia, and Urea Cycle Disorders (UCD) are known to present as metabolic encephalopathy and hence metabolic encephalopathy was the most common symptom in our study. As seen in study by Kamate et al., maximum number of cases was organic acidemias . In our study, 32 patients (87.5% patients) were less than 2 years, 6 out of them were neonates (18.75%). In study by, Sivaraman et al.  almost half have been diagnosed in the neonatal period itself, which is most likely to indicate a more severe spectrum of IEM. This could be due to early suspicion of team of NICU in this study. Amongst organic acidemias, glutaric academia II (2), propionic academia (2), methylmalonic acidemia (2), glutaric acidemia (1), MSUD (1), Biotin responsive basal ganglia disease (1) were the common organic acidemias which can be picked up on extended newborn screening. A pilot study in India also identified these as common organic acidemias in newborn screening .
Amongst the rare organic acidemias diagnosed based on TMS as a screening and GCMS as a gold standard test and genetic study in few, were 2 methyl 3 hydroxy butyric aciduria, 2- hydroxy Glutaric aciduria, Oxoprolinuria, 3 methyl glutaconic aciduria type 5, HMG coA lyase deficiency and riboflavin deficiency etc. L-2 hydroxy glutaric aciduria has been reported in India by Kamte et al.  and Balaji et al. . Oxoprolinuria has been reported in by Bhaskaranand et al.  in a pediatric patient and an adult patient by Senthilkumaran et al. . A 7-month old girl child with metabolic encephalopathy was diagnosed with 2 methyl 3 hydroxy butyric aciduria which is an X linked dominant rare IEM and not reported in India. 21 days old male child was admitted with metabolic encephalopathy, was diagnosed with HMG CoA lyase deficiency, which is a defect in ketogenesis.
This is not reported in India and has been reported by Sass et al.  2-year-old male child with global developmental delay was diagnosed with 3 methyl glutaconic aciduria type 5 based on homozygous likely pathogenic variants in exon 5 of DNAJC19 gene c.250C>T (pArg84Ter). To date, maximum cases of DCMA reported involve individuals from the Dariusleut Hutterite population, an endogamous population of the Great Plains region of Canada and the northern United States. Update on cases, natural history by Machiraju et al. has described phenotypes in this disease . This patient had global developmental delay with ataxia and micropenis requiring testosterone injections in infancy.
2D echo was normal. Another 2 years old child with global developmental delay, movement disorder and seizure disorder had TMS and GCMS negative but exome sequencing revealed homozygous likely pathogenic variants in SLC19A3 gene in exon 3, c.595 T>A causing biotin responsive basal ganglia disease. There is a case series by Majid et al. , Kassem et al.  and 3 case reports from India [22-24]. A 9-day old female succumbed to metabolic encephalopathy and TMS was screen positive for MSUD. Carrier screening of parents by next generation sequencing revealed that both parents were carrier of heterozygous likely pathogenic variant in BCKDHA gene in exon 9 c.1251delC.
There is a case series by Bashyam et al. and Narayan et al. from India [25,26]. 3-day old male child succumbed to encephalopathy that also had similar sibling death on day 3 of life, was screen positive for methylmalonic academia. His parents were having compound heterozyous likely pathogenic variants in MMUT gene c.643G>T(pGly215Cys), and second variant c.692dup, pTyr231Ter. Observed variants are already reported in literature. 2 patients with glutaric academia II, 1 patient with glutaric acidemia I, 1 patient with riboflavin deficiency, 1 with 3 methyl glutaconic aciduria types 5 are on regular follow up with dietary modifications and supplements. Amongst the common aminoacidopathies, 2 patients were diagnosed with tyrosinemia type 1 and 2 with hyperphenylalaninemia, 1 with hyper-homocystenemia and 1 with a rare amino acid disorder of lysinuric protein intolerance. We have already reported the case of lysinuric protein intolerance . One patient with hyperphenylalaninemia underwent urinary pterin assay and was suspected to be suffering from biopterin pathway defect. His clinical exome analysis revealed homozygous likely pathogenic variant in exon 4, c.200C>T (pThr67Met) in PTS gene, causing Hyperphenylalaninemia, BH4-deficient, A. There is a case series and a case report from India on BH4 deficient hyperphenylalaninemia [28,29].
Amongst urea cycle defects, all 3 patients were diagnosed with citrullinemia, two patients were diagnosed in neonatal age with encephalopathy and third child was diagnosed late with behavioral changes. Citrullinemia was common UCD in latest study from India . Molecular analysis in one patient revealed common mutation of c.1168G> A (pGly390Arg) as described in the study by Bijarniya et al. Amongst the fatty acid oxidation defects, acyl carnitine profile in TMS, was suggestive of 3 patients with carnitine uptake defects, 2 patients with long chain acyl Co A dehydrogenase deficiency (LCHAD), one each with short chain and medium chain acyl Co A dehydrogenase deficiency (SCAD and MCAD). Patients with carnitine uptake defects, MCAD deficiency presented with symptoms of hypoglycemia, hepatomegaly and raised liver enzymes and deranged PT INR.
All patients are under treatment with reduced episode of hypoglycemia following precautionary advice. Patients with SCAD and LCHAD deficiency presented with metabolic encephalopathy with seizure disorder and succumbed to acute encephalopathy. All the FAODs in our study have low prevalence in India with few case reports [31-33]. One patient was diagnosed with neurotransmitter metabolic defect of GABA (gamma amino butyric acid), a girl child with global developmental delay and autistic features. She was diagnosed with c.701 C>T homozygous likely pathogenic variant in ALDH5A1 gene. SSADH deficiency has been reported in India from two studies [34,35]. Approximately 450 cases are diagnosed with SSADH deficiency worldwide .
One child was diagnosed with purine metabolic defect. 17 month old child with global developmental delay was diagnosed with inosine triphosphate phosphohydrolase deficiency, based on homozygous pathogenic variants in ITPA gene, c.137delA (pGln46ArgfsTer43). Homozygous or compound heterozygous mutations in ITPA gene are known to cause neurological presentations. One case reported by Karthik et al.  has similar features of encephalopathy, global developmental delay and MRI brain abnormalities. Another study by Nicholas has described the similar phenotype . TMS and GCMS was screen positive for galactosemia as in our case of 2 months old girl child who presented with hepatic failure, hypoglycemia and bilateral cataract, her DNA study revealed compound heterozygous pathogenic variants c.142 C>T and c.610 C>T in GALT gene. There are case series and newborn screening studies on galactosemia in India [39-42].
Amongst the 4 patients with mitochondrial disease, all 4 had typical MRI brain findings of symmetrical T2 weighted hyper intensities in bilateral, basal ganglia, midbrain, pons and cerebellum and lactate peak on MRS. A 2-year-old girl with motor delay and optic atrophy underwent molecular testing and compound heterozygous variant in TWNK gene, one heterozygous likely pathogenic variant c.1003 C>A and a second variant of unknown significance c.2050 A>C causing mitochondrial DNA depletion syndrome 7 was detected. All individuals with the IOSCA founder variant in TWNK have been identified in the genetically isolated population of Finland only, where IOSCA is the second-most common inherited ataxia . Other TWNK variants have been described in affected individuals of English, Pakistani, Indian origin [44-46]. In developed countries, newborn screening is being done widely for varying metabolic disorders. The conditions screened are 6, 29, and 23 conditions, in the UK, USA, and Australia, respectively . Among the patients who presented for the first time, more than 50% patients could have been potentially picked up by NBS.
Molecular testing was done in 13 cases (32.5%) of the study cohort. We could not confirm using molecular analysis in a higher proportion and had to do parental carrier screening by clinical exome studies in few. It is a good practice to store DNA even when the testing cannot be done at an acute state during NICU or PICU admission. As all the conditions in the present study are inherited in an autosomal recessive manner, we got 72% (8/11) of a pathogenic variant in homozygous status and only 23% (3/13) as compound heterozygous. In India due to the high prevalence of consanguineous marriage and endogamous population there is a high chance of these conditions occurring in the homozygous state rather than the compound heterozygous state. In the present genomic era, next-generation sequencing is the most common test utilized to detect single gene disorder exome sequencing is an effective technology for diagnosing metabolic disorders. Molecular analysis has not only helped the index child but also in prenatal diagnosis in 3 families and fetuses were carrier for the disease.
As this study describes only the short-term outcome, it is not taking into account the death that could potentially happen outside the study period or which could happen at a different hospital or home. This is one of the limitations of the study. Strengths of our study are a detailed description of biochemical testing aiding the definitive diagnosis and diagnosing rare IEMs and mutations unique to our region.
TMS and urine GCMS is helpful in facilitating early diagnosis and timely treatment of inherited metabolic disorders. Because of high degree of consanguinity and marriages in same community, common as well as many rare inherited metabolic diseases were diagnosed. Clinico-etiological profile study has thrown light on clinical features, natural course of many common and rare IEMs and it may provide clinicians with a deeper understanding of these conditions, allowing for improved early diagnosis and treatment of these diseases. By creating this data resource we aimed to leverage an overview of different common and rare IEMs found in our region, and document the genetic variants that are relevant to the diagnosis of IEMs.
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