Ayesha Hafeez  ( Armed Forces Institute of Pathology, Rawalpindi )
Aamir Ijaz  ( Rehman Medical Institute, Peshawar )
Nayyar Chaudhry  ( Armed Forces Institute of Pathology, Rawalpindi )
Oshaque Ali  ( Armed Forces Institute of Pathology, Rawalpindi )
Muhammad Tahir Khadim  ( Armed Forces Institute of Pathology, Rawalpindi )
Muhammad Tahir Khadim  ( Armed Forces Institute of Pathology, Rawalpindi )

Objective: To determine the pattern of various inherited metabolic disorders specifically through plasma amino acid and urine organic acid testing in high-risk paediatric population.

Method: The cross-sectional retrospective study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, and comprised data from April 2015 to March 2018 of children referred to the Department of Chemical Pathology and Endocrinology for work-up of suspected inherited metabolic disorders. Complete clinical history, baseline biochemical investigations, plasma amino acid and urine organic acid profiles, where indicated, were collected. Quantitative plasma amino acid and analysis was carried out by Ion Exchange Chromatography on Biochrome 30+ amino acid analyser, and urine organic acid analysis by Gas Chromatography-Mass Spectrometry. Findings were linked to the identified disorders. SPSS 21 was used for data analysis.

Results: Of the 805 cases reviewed, 49(6%) had an inherited metabolic disorder. Male:Female ratio of the cases was 1.5:1, and the median age was 240 days (interquartile range: 1-15695 days). The most common presenting symptom was seizures 316(39.3%) followed by lethargy 283(35.2%). Of the diagnosed cases, aminoacidopathies were 28(57%) and in them, non-ketotic hyperglycaemia accounted for 7(25%.). There were 12(24.5%) cases of organic acidurias followed by 9(18.4%) that were other than the two diagnoses.

Conclusion: The cases of inherited metabolic disorder detected indicated significant prevalence. Non-ketotic hyperglycinemia was the commonest disorder diagnosed.

Keywords: Ion Exchange Chromatography, Aminoacidopathies, Inherited Metabolic Disorders, Organic Aciduria. (JPMA 70: 53; 2020). https://doi.org/10.5455/JPMA.301908

Introduction

Although individually rare, the aggregate incidence of Inherited Metabolic Disorders (IMDs) may approach 1 in 1,000 newborns.¹ In a country like Pakistan, where marriages between cousins have prevailed for centuries and the rate of consanguinity is high, ² there is an increased risk of inheriting various genetic disorders. In healthcare setups facing resource-constraints and limited technical expertise, the diagnosis of IMDs poses a real challenge. Diagnosing these disorders is not always very straightforward and involves a multi- disciplinary approach. The most difficult task for a clinician is to know when to consider IMD and which tests to order for evaluation. ³ The signs and symptoms for various disorders are non-specific⁴ and, moreover, other diagnoses like sepsis, anoxic ischaemic encephalopathy (in neonates), pulmonary haemorrhage etc., may accompany IMDs which makes the diagnosis even more challenging. In any case, the optimal outcome for these children depends upon early recognition of the signs and symptoms, expert evaluation, and prompt referral to a centre familiar with the evaluation and management of these disorders. Another stumbling block in diagnosis is the need for the exclusion of routine childhood illnesses in the first place, unfamiliarity with biochemical interrelationships⁵ and inappropriate sample collection or storage. In all cases, a battery of certain routine biochemical investigations need to be carried out before undertaking the more sophisticated investigations, which compounds the expenses involved. The specific metabolite testing includes Amino acid (AA) and urine organic acid (OA) analysis which helps in diagnosing mostly aminoacidopathies as well as organic acidemias. Plasma AA analysis by ion exchange chromatography (IEC) is a state-of-the-art technique for the quantitative analysis of AAs in various body fluids like plasma, urine and cerebrospinal fluid (CSF). Multiple AAs and their metabolites can be analysed in each run. Likewise, urine OA analysis on gas chromatography mass spectrometry (GCMS) is an excellent state-of-the-art technique for detecting various organic acidemias. Final confirmation relies on enzyme and genetic analyses. ⁶ There is no programme available on the national level for the diagnosis of IMDs and only a few centres in Pakistan are currently undertaking testing for specific metabolites like AAs and OAs for IMD testing. The current study was planned to determine the frequency and pattern of IMDs detected in our setup.

 

Materials and Methods

The cross-sectional retrospective study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, and comprised data from April 2015 to March 2018 of patients referred from the paediatric outpatient department (OPD) and neonatal intensive care unit (NICU) to the Department of Chemical Pathology and E ndocrinology for work-up of suspec ted IMD s. Blood samples were collected for basic biochemical and specialised tests. Ammonia levels were collected in lithium heparin tube, and plasma lactate / glucose in sodium fluoride containers. Urine was collected in plain containers for non-glucose-reducing substances and q ualitative meta bolic screening ferricchloride ^2,4 - Dinitrophenylhydrazine (DNPH), cyanidenitroprusside and test for ketones. Blood complete picture (CP) was done on Sysmex Haematology analyser, while plasma ammonia, lactate and liver function tests (LFTs) were done on Clinical Chemistry Analyzer (Advia 1800-Siemens, Germany). For plasma AA analysis, 2-3ml blood was collected in lithium heparin tubes and transported on ice to laboratory. The plasma was separated immediately by centrifugation at 3500 rounds per minute (rpm) for 5 minutes and frozen in aliquots at -80°C if there was a delay in analysis. AA analysis was carried on biochrome 30+ AA analyser (Biochrome® UK) based on ion exchange chromatography using lithium column (4.6mm diameter) for separation. Further, 5% sulfo-salicylic acid was added and the contents were allowed to stand for 30 minutes at 40C, followed by vortex centrifugation and filtration through a 0.2μm membrane. The filtrate was taken in sample vials to the analyser. Ninhydrin solution was utilised and after post-column derivatisation, the amount of colour produced was measured which was directly proportional to the quantity of AAs present in the sample. Coloured compounds were quantified at 440nm for hydroxypoline and proline, and at 570nm for the rest of the AAs. Individual AAs were detected based on their respective retention times, quantified by the area under the peak, and results were analysed by Open Lab control panel software version (Agilent Technology). Quality control was maintained in each run with 2 levels of controls (ClinChek® Recipe), with inter-assay and intraassay coefficient of variation (CV) <1%, and L-norleucine (100mg; Sigma Life sciences) was utilised as the internal standard (ISD). All reagents were prepared using highperformance liquid chromatography (HPLC) grade water. Urine OA screening was performed on GCMS (Agilent Technologies, USA, 7890A GC system, with 5975C inert Mass Selective Detector). For the purpose, 10ml of firstmorning- voided urine was obtained in a plain container. Urine creatinine (mmol/L) was measured in all samples on Clinical Chemistry Analyser (Advia 1800-Siemens, Germany). Liquid-liquid extraction was utilised to extract OA from the samples, followed by oximation, extraction and derivatisation. 3,3 dimethylglutaric acid (Sigma Aldrich) was utilised as the ISD. Results were accepted when the abundance of ISD in the chromatogram was >1.0 x 106. For the diagnosis of phenylketonuria (PKU) we used a plasma phenylalanine (Phe) to tyrosine (Tyr) (Phe-to-Tyr) ratio of >37 and for diagnosis of hyperphenylalanemia (HPA), a Phe cut-off of <1200 mol/L8 was used in addition to the aforementioned ratio. Non-ketotic hyperglycinemia (NKH) diagnosis was based on raised CSF-to-plasma ratio of glycine (>0.02)9 performed on parallel samples, in the absence of valproate therapy or a significant urine OA profile. Type 1 tyrosinemia was diagnosed based on clinical features, disturbed LFTs, markedly raised Tyr levels on plasma AA analysis and marked qualitative elevations of succinylacetone in urine by GCMS. Maple syrup urine disease (MSUD) ¹⁰ was diagnosed based on markedly elevated concentrations of branched chain AA leucine, isoleucine and valine and elevated concentration ratios of these AAs to others. Data was analysed using Mass Hunter software utilizing Orgasid library¹¹ A questionnaire comprising complete clinical and biochemical data was filled out for all the samples. Results were analysed using SPSS 21. Data was expressed as mean and standard deviation (SD) for quantitative variables, and frequencies and percentage for qualitative variables.

Results

Of the 805 cases reviewed, 49(6%) had an inherited metabolic disorder. Male:Female ratio of the cases was 1.5:1, and the median age was 240 days (interquartile range [IQR]: 1-15695 days). Rate of consanguinity was 563(70%). The most common presenting symptom was seizures 316(39.3%) followed by lethargy 283(35.2%) (Table 1).





Age was divided into three categories 0-1 year 29(59%) cases, 1-5 year 14(28%) cases and 5-11 years 6(13%) cases. Of the diagnosed cases, 38(78%) were received from intensive care setups, 4(9%) from nonintensive, and 6(13%) from the OPD. Seizure was the commonest presenting feature in children <1 year of age 11(39%), while delay in developmental milestones was the commonest feature among those aged >1 year 10(20%). Raised ammonia levels 232.2±65.8 and disturbed LFTs, mainly raised bilirubin, were the most striking biochemical derangements ( Tab l e 2 ) . Out of 49 diagnosed IMD cases, 28(57%) had aminoacidopathies and 12(24%) had organic acidurias. Among the aminoacidopathies, there were 4(14.3%)HPAs, 3(10.7%)PKUs, 7(25%) NKHs, 2(7%) hyperprolinemia cases, 5(18%) cases of tyrosinaemia Type 1, 4(14.3%) urea cycle defects, and 3(10.7%) cases of homocystinuria. Organic acidurias included 3(25%) cases of glutaric aciduria Type 1, 4(33.3%) methylmalonic aciduria, 2(16.6%) propionic academia and 3(25%) cases of maple syrup urine disease (MSUD). Cases diagnosed with disorders other than aminoacidopathies and organic aciduria were 6(12%); 1(16.6%) each of glycogen storage disease (GSD), Fanconi syndrome, and congenital lactic acidosis, and 3(50%) alkaptonuria. Finally, 2(4%) cases were suspected of having mitochondrial disorder, and 1(2%) of having pyruvate carboxylase deficiency. The prevalence of IMDs  in children aged <1 year was the highest (Figure).



Of the total specimen, 45(5.5%) were either recalled or repeated for various reasons, such as non-availability of clinical data 22(2.8%), gross haemolysis 11(1.2%), or icterus 12(1.5%). Hyperbilirubinaemia was the most common analytical challenge, whereby we found analytical interference in the form of multiple peaks around retention times of some of the AAs .

Discussion

The data presented by the current study is unique in the sense that it belongs to one of the few centres in Pakistan equipped with facilities for testing specific metabolites like AA and OA for diagnosing IMDs. In most of the local data presented, these tests have been outsourced abroad. ^12,13 Our results have shown a significant proportion of patients (14.2%) who were diagnosed as NKH which is in accordance with literature. Only one patient was on valproate therapy, but at the time of sampling he had been off-treatment for at least 4 weeks. Most of the cases diagnosed were aged <6 months. We also diagnosed a rare presentation of IMD in a child who had seizures in the 8th year of life and later investigations revealed encephalopathy due to atypical NKH. ¹⁴ Measurement of glucosylceramide synthase (GCS) enzyme activity in liver biopsy specimen ¹⁵ is an invasive procedure and molecular testing facility was not available in our centre so we used the raised CSF-to-plasma glycine levels in all patients . Out of the five cases of type 1 tyrosinemia diagnosed, one of the cases had a full-blown renal involvement with phosphaturia, amino aciduria and glycosuria. Another was a case of 9-year-old child presenting with rickets and cirrhosis and the plasma AA analysis showed elevated Tyr and Phe levels along with moderate elevation of methionine levels. This AA profile was similar to one case reported in a study. ¹⁶ Two of the five children we diagnosed had markedly elevated alpha fetoprotein levels. This biochemical finding was similar to that reported earlier. ¹⁷ For diagnosis of PKU, in our laboratory, we used a plasma Phe-to-Tyr ratio of >3.For diagnosis of HPA, a Phe cutoff of <1200 mol/L was used in addition to the ratio described earlier. MSUD was diagnosed based on markedly elevated concentrations of branched chain amino acids leucine, isoleucine and valine and elevated concentration ratios of these AAs to others. CSF leucine and isoleucine levels were also markedly elevated in all cases much in accordance with literature. ¹⁸ Mitochondrial disorders were suspected in 2 patients on the basis of clinical history, examination, biochemical findings and findings of brain MRI. In the current study, 49(6%) cases were diagnosed as IMDs. Data from Pakistani population is still limited. The prevalence of IMDs in our study closely matched that reported by an earlier study by Sherazi NA et al. ¹¹ Seizures and coma were the most common presentations in our study, and that also matches literature. ¹⁹ We did not find a very high frequency of storage disorders in patients referred to us. This contrasts with a study which found a much greater frequency of GSDs and Gaucher disease compared to other IMDs. ²⁰ This difference in prevalence can be explained keeping in mind the fact that the data of earlier study was from Gastroenterology, Hepatology and Nutrition departments of a paediatric hospital. A study showed a high rate of consanguinity²¹which is a finding similar to that seen in our study (70%). Similarly positive history of a previously affected sibling was 35.5%, much in accordance with our study. It was seen that aminoacidopathies like NKH, type 1 tyrosinaemia and various organic acidurias encompassed most of the high-risk cases which, again, is in accordance with literature. ²² We faced several analytical challenges during the course of workup of these cases referred for suspected IMDs. A total of 45 samples were either recalled or repeated for various reasons such as non-availability of clinical data, presence of gross haemolysis or icterus. Haemolysis yielded rise in multiple amino acids like glycine, serine, lysine, glutamic acid and taurine which made the  interpretation difficult. Arriving at a provisional diagnosis for any IMDs needs a constellation of biochemical resultsrather than a single investigation. A report of plasma AAs can be meaningfully interpreted in the presence of relevant clinical and biochemical data. This aspect was taken care of by close collaboration with the treating/referring paediatrician. The samples with gross haemolysis were not analysed and recalled. This is due to the fact that haemolysis may cause increases in certain AAs like serine, glycine, taurine, aspartic acid, glutamic acid, ornithine and decreased arginine. Moreover, haemolysis, along with certain other factors is found to influence sample stability. ^22,23Bilirubin remained the most common analytical challenge. ^23,24 Analysis was recommended to be carried out/repeated after bilirubin levels had settled. Some of the cases were regularly followed-up. Four out of the seven cases diagnosed as NKH expired despite the initiation of treatment. One of the cases diagnosed as atypical NKH was managed during admission with anti-epileptics to which seizures responded but abnormal movements settled partially. Oral folic acid and vitamin B12 were also added to his medications. His condition had improved on discharge and he was advised regular follow-up, and genetic counselling sessions were carried out with the parents. ²⁵ Another 5-year-old diagnosed as homocystinuria was started on pyridoxine, folic acid and a low-methionine diet. However, he showed no biochemical response to pyridoxine and was advised to continue on low-methionine and high-cysteine diet. ²⁶ Likewise, another case diagnosed with glutaric aciduria was advised to be kept on low-protein and low-lysine diet along with carnitine nutritional supplementation with further advice to parents to ensure avoidance of prolonged fasting intervals. Two of the children diagnosed with MSUD expired during admission while the third 24-days-old baby was managed initially on antibiotics, intravenous (IV ) fluids, multivitamin supplements and mechanical ventilation. He was started onleucine – free milk powder on discharge.

 

Conclusion

The 6% prevalence of IMDs was quite significant. NKH was the commonest IMD diagnosed. IMDs must be kept in mind while treating a sick child in parallel with sepsis and a properly worked out criteria will be quite useful in the selection of infants to undergo expensive and sophisticated investigations like molecular testing.

Disclaimer: None.

Conflict of interest: None.

Source of Funding: None.

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