Biochemical Genetics Laboratory Research Articles

Clinico-Biochemical Spectrum of Pakistani Patients with Glutaric Aciduria Type 1 (GA1): Experience from a Specialised Biochemical Genetics Laboratory in Pakistan.

To evaluate the clinical, radiological, and biochemical features of glutaric aciduria Type 1 (GA1) patients identified through urine organic acid testing at a biochemical genetics laboratory (BGL) in Pakistan. Observational study. Place and Duration of the Study: Department of Pathology and Laboratory Medicine, The Aga Khan University Hospital, Karachi, Pakistan, from January 2013 to December 2022. Medical charts and urine organic acid (UOA) chromatograms of the patients presenting at the BGL from January 2013 to December 2022 were reviewed. Brain imaging was obtained where available. Variables were noted as per the objective and descriptive statistics were obtained. GA1 was found in 64 (0.4%) patients out of a total of 16,094 UOA requests for high-risk screening cases. The age of diagnosis ranged between one month and three years. The brain MRI findings revealed characteristic abnormalities such as cerebral atrophy, expanded CSF spaces, white matter abnormalities, and a distinct bat wings appearance, in cohesion with the results of biochemical testing. Sixty-four cases of GA1 from a single centre indicate a high frequency of the disorder in Pakistan. Late diagnosis emphasises the need for increased clinical awareness and preferably newborn screening to ensure optimal outcomes. Glutaric aciduria Type 1 (GA1), Brain imaging, UOA analysis, Glutaryl-CoA dehydrogenase (GCDH), Pakistan.

Clinical and biochemical spectrum of propionic Acidemia – Experience from a biochemical genetics laboratory in Pakistan

Clinical and biochemical spectrum of propionic Acidemia – Experience from a biochemical genetics laboratory in Pakistan

Validating urinary orotic acid through high-pressure liquid chromatography (HPLC): Insights from a specialized biochemical genetics laboratory in Pakistan

Validating urinary orotic acid through high-pressure liquid chromatography (HPLC): Insights from a specialized biochemical genetics laboratory in Pakistan

B-161 Mass Spectrometric Assay for the Early Diagnosis of Barth Syndrome

Abstract Background Barth syndrome (BTHS) is an inborn error of phospholipid metabolism caused by pathogenic variants in the TAFAZZIN gene locus on a chromosome X. TAFAZZIN encodes a phospholipid transacylase enzyme, tafazzin, that promotes cardiolipin (CL) remodeling. Deficiency of tafazzin activity in BTHS results in accumulation of monolysocardiolipin (MLCL) and decreased abundance of mature CL, making an increased ratio of MLCL to CL as a diagnostic marker for BTHS. 52:2-MLCL to 72:8-CL in dried-blood spot (DBS) has been used for provisional identification BTHS using LC-MS/MS. A development of multiplexed method that targets several species of MLCL and CL can enhance the diagnosis of BTHS. Methods A targeted LC-MS/MS with scheduled multiple reaction monitoring (MRM) mode in negative electrospray ionization was developed using a triple quadrupole MS. DBS from patients with BTHS were obtained at Johns Hopkins University and provided by Barth Syndrome Foundation while control DBS were provided by Biochemical Genetics Laboratory at Mayo Clinic. Internal standards and chloroform:methanol (1:1, v/v) were added to DBS (1/8″ diameter) and sonicated. Supernatants were collected and dried under nitrogen gas. Followed by reconstitution in methanol, samples were subjected to LC-MS/MS analysis. Results More than 20 species of MLCL and CL species having various fatty acyl chains were detected and quantified. Remodeled CL species were decreased while MLCL and nascent CL species were found to be increased in known patients of BTHS (n = 8) compared to controls (n = 8). Notably, several ratios of lipids including 52:2-MLCL/72:8-CL and 54:2-MLCL/70:6-CL showed >300-fold elevation in the patients. Conclusion Various species of MLCL and CL that can be used to diagnose BTHS can be measured from an easily accessible specimen such as DBS using this method.

Impact of Structural GLA Protein Changes on Peripheral GLA Activity and Substrate Accumulation in Fabry Disease Patients.

Fabry disease is an X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, leading to decreased/absent α-galactosidase activity. In clinical practice, enzyme activity and substrate/byproduct accumulation play a role in diagnosis and disease-monitoring biomarkers. However, interpreting biomarker levels is not straightforward and can change according to the underlying GLA protein abnormality. Our goals were to understand how disrupting specific protein regions changes biomarker behaviour and to establish specific patterns for individual variants. We analysed data from the Biochemical Genetics Laboratory regarding GLA variants, GLA enzyme activity (in dried blood spots, plasma or white blood cells), plasma LysoGb3 accumulation, and urinary Gb3 excretion. We assessed correlations, trends, and potential predictor models of biomarker behaviour. We assessed 169 hemizygous male and 255 heterozygous female patients. For both groups, substrate accumulation correlates inversely with GLA activity. Variants affecting residues buried within the protein core or the active site were associated with more severe biomarker changes, while those affecting residues that establish disulfide bonds or are glycosylated were similar to other variants. For each non-truncating variant, we also established specific profiles of biomarker behaviour. Finally, we also designed predictor models of biomarker behaviour based on structural variant information. This study provides the groundwork for the impact of GLA protein variation on GLA activity and substrate accumulation. This knowledge is of extreme relevance for diagnostic labs and clinicians, as some genetic variants are challenging to interpret regarding pathogenicity. Assessing whether biomarker changes are in the expected range for a specific variant may help diagnostic evaluation. This study also contributes to recognising non-disease-causing variants, considering their overall biochemical impact, and providing a comparative reference for biomarker discovery studies. In the future, the correlation of these findings with disease severity may be of great relevance for diagnosis and monitoring progression.

P14.05.B 2-HYDROXYGLUTARATE IN CEREBROSPINAL FLUID AS A MONITORING BIOMARKER FOR IDH-MUTANT GLIOMAS

Abstract BACKGROUND D-2-hydroxyglutarate (D-2-HG) is a well-established oncometabolite of isocitrate dehydrogenase (IDH) mutant gliomas. While prior studies have demonstrated that D-2-HG is elevated in the cerebrospinal fluid (CSF) of patients with IDH-mutant gliomas, no study has evaluated the ability of CSF D-2-HG to quantitatively monitor tumor burden and therapeutic response. Toward this goal, we are routinely obtaining CSF samples from patients prior to, during, and after treatment via intra-operative collection and CSF-access devices. MATERIAL AND METHODS Patients were enrolled into our Ommaya reservoir study (NCT04692337), which enables longitudinal access to CSF. The catheter for the implanted reservoir was placed within the surgical resection cavity following a standard-of-care resection. Intra-operatively collected CSF samples were also biobanked through Mayo Clinic’s Neuro-Oncology biorepository. CSF samples were also acquired via clinically indicated ventriculoperitoneal shunts (VP shunts) or external ventricular drains via our ongoing CSF biomarkers study (NCT04692324). D-and-L-2-HG were assayed from each CSF sample via gas chromatography-mass spectrometry (GC-MS) at Mayo Clinic’s Biochemical Genetics Laboratory. RESULTS CSF was collected prior to and after resection in patients via Ommaya reservoirs or VP shunts, all of whom underwent near gross total resections (>95% of tumor volume). D-2-HG and the D/L ratio fell on average by 57.9% (range: 15.1%-98.6%) and 68.8% (range: 49.3%-98.9%), respectively, following resection. In one patient with a largely intraventricular grade 4 IDH-mutant astrocytoma, D-and-L-2-HG fell by 99.1% and 99.3% by post-operative day 2, consistent with the impact of ventricular contact on baseline CSF D-2-HG levels. In patients for whom CSF was collected throughout chemoradiation, with or without immunotherapy, D-2-HG and the D/L ratio continued to decrease over time, despite concern for pseudoprogression versus progression on imaging. CONCLUSION Our early experience with this strategy demonstrates the potential for intracranial CSF D-2-HG as a monitoring biomarker for IDH-mutant gliomas. Work is ongoing to evaluate the reproducibility of these findings in a larger cohort of patients with low or high-grade gliomas, in addition to the impact of tumor recurrence on 2-HG. Future studies will evaluate the impact of IDH inhibitors and other experimental therapies on CSF 2-HG levels for pharmacodynamic monitoring of treatment response.

Optimization of the biochemical genetics laboratory rotation using a multidesign approach to curriculum

PurposeA biochemical genetics laboratory rotation is required for multiple genetics training programs. Traditionally, this rotation has been observational with experience being dependent upon cases released and availability of laboratory director(s), resulting in inconsistent learning opportunities. This curriculum was created to standardize the learning experience. MethodsThe revised rotation provides multiple teaching modalities including small group didactic sessions (flipped classroom model), case-based sessions, and hands-on laboratory experience. Trainees prepare a presentation (learning by teaching) and discuss the differential diagnosis, metabolic pathway, newborn screening, treatment, and molecular characteristics of the gene(s) implicated. Learner assessment is performed using pre- and post-tests, learner evaluations, and instructor feedback. ResultsPre- and post-test scores were significantly different (P < .001) for learners from all programs. Participants found the course to be effective, increased their learning, and allowed them to interact with metabolic testing results in helpful ways. Faculty appreciated the use of prerecorded lectures and additional time for in-depth teaching on interesting cases. ConclusionThe revised rotation has been well received by trainees and faculty. Interaction of learners with the laboratory staff was optimized by ensuring all parties were prepared to teach and learn. Future directions include expanding the program to include remote learners from other centers.

Biotin-responsive Multiple Carboxylase Deficiency (MCD).

This study aimed to determine the clinical spectrum and biochemical findings on urine organic acids (UOA) in Biotin-responsive multiple carboxylase deficiency (MCD) patients presenting to the biochemical genetics laboratory (BGL). Patients reported as MCD, from January 2013-December 2020 were included. The UOA was analysed by gas chromatography mass spectrometer. Demographic, clinical, and biochemical details were extracted from the BGL history form. Two hundred and two patients were reported to have biotin responsive MCD with 111(55%) males and a median (Q3-Q1) age of 7 months (13-4). Of these 71.7% (n=145) patients presented in infantile period. Parental consanguinity was observed in 80% (n=161) and another 32.6% (n=66) cases grandparents were cousins. The main presenting features were seizures, developmental delay, and lethargy. The common peaks were determined on UOA 3OHIVA, MC and MCC. MCD is not rare in Pakistani population; it is recommended to include this disorder in newborn screening programs. Key Words: Biotin responsive multiple carboxylase deficiency, Organic acids, Amino acids, Pakistan, Inborn errors of metabolism.

A simple method modification to increase separation of 2- and 3-hydroxyglutaric acid by GC–MS for clinical urine organic acids analysis

Urine organic acids profiling by gas chromatography-mass spectrometry (GC–MS) is routinely performed in hospital biochemical genetics laboratories for the investigation of inborn errors of metabolism. In particular, accurate identification of urinary levels of 3-hydroxyglutaric acid (3-OHGA) is important for diagnosing glutaric aciduria type 1 (GA1), but can be challenging by routine GC–MS profiling analysis due to co-elution and spectral similarity with the isomer 2-hydroxyglutaric acid (2-OHGA). To improve analytical specificity, unique ions were selected and a simple second-tier reinjection method was developed to enhance the chromatographic separation of the 2- and 3-OHGA isomers and potential unknown interferences. Specimens flagging on the routine analysis were simply reinjected on the same GC column using a modified temperature gradient containing an isothermal hold. Correlation between the reinjection and initial methods was higher for 2-OHGA (R = 0.9612) compared to 3-OHGA (R = 0.7242). Mean differences between the reinjection and initial methods for 2-OHGA and 3-OHGA were −8.5% and −61.1% respectively. The large decrease in 3-OHGA concentration for many specimens using the reinjection method was primarily attributable to separation from unknown variable interference(s) that were falsely elevating 3-OHGA in the initial analysis despite the use of a more unique quantifier ion. Overall, the reinjection approach increased analytical specificity in evaluating for the presence of increased urinary 3-OHGA. This second-tier approach, using a GC isothermal hold, could easily be implemented or adapted by other clinical laboratories experiencing related diagnostic challenges.

Successful Implementation of Expanded Newborn Screening in the Philippines Using Tandem Mass Spectrometry.

Newborn bloodspot screening (NBS) began as a research project in the Philippines in 1996 and was mandated by law in 2004. The program initially included screening for five conditions, with a sixth added in 2012. As screening technology and medical knowledge have advanced, NBS programs in countries with developed economies have also expanded, not only in the number of newborns screened but also in the number of conditions included in the screening. Various approaches have been taken regarding selection of conditions to be screened. With limited resources, low- and middle-income countries face significant challenges in selecting conditions for screening and in implementing sustainable screening programs. Building on expansion experiences in the U.S. and data from California on Filipinos born and screened there, the Philippine NBS program has recently completed its expansion to include 29 screening conditions. This report focuses on those conditions detectable through tandem mass spectrometry. Expanded screening was implemented in a stepwise fashion across the seven newborn screening laboratories in the Philippines. A university-based biochemical genetics laboratory provides confirmatory testing. Follow-up care for confirmed cases is monitored and provided through the NBS continuity clinics across the archipelago. Pre-COVID-19 pandemic, the coverage was 91.6% but dropped to 80.4% by the end of 2020 due to closure of borders between cities, provinces, and islands.

Bile Acid Profiles in Primary Sclerosing Cholangitis and Their Ability to Predict Hepatic Decompensation.

Altered bile acid (BA) homeostasis is an intrinsic facet of cholestatic liver diseases, but clinical usefulness of plasma BA assessment in primary sclerosing cholangitis (PSC) remains understudied. We performed BA profiling in a large retrospective cohort of patients with PSC and matched healthy controls, hypothesizing that plasma BA profiles vary among patients and have clinical utility. Plasma BA profiling was performed in the Clinical Biochemical Genetics Laboratory at Mayo Clinic using a mass spectrometry based assay. Cox proportional hazard (univariate) and gradient boosting machines (multivariable) models were used to evaluate whether BA variables predict 5-year risk of hepatic decompensation (HD; defined as ascites, variceal hemorrhage, or encephalopathy). There were 400 patients with PSC and 302 controls in the derivation cohort (Mayo Clinic) and 108 patients with PSC in the validation cohort (Norwegian PSC Research Center). Patients with PSC had increased BA levels, conjugated fraction, and primary-to-secondary BA ratios relative to controls. Ursodeoxycholic acid (UDCA) increased total plasma BA level while lowering cholic acid and chenodeoxycholic acid concentrations. Patients without inflammatory bowel disease (IBD) had primary-to-secondary BA ratios between those of controls and patients with ulcerative colitis. HD risk was associated with increased concentration and conjugated fraction of many BA, whereas higher G:T conjugation ratios were protective. The machine-learning model, PSC-BA profile score (concordance statistic [C-statistic], 0.95), predicted HD better than individual measures, including alkaline phosphatase, and performed well in validation (C-statistic, 0.86). Patients with PSC demonstrated alterations of plasma BA consistent with known mechanisms of cholestasis, UDCA treatment, and IBD. Notably, BA profiles predicted future HD, establishing the clinical potential of BA profiling, which may be suited for use in clinical trials.

A Review of the Results of the Very Long Chain Fatty Acid Analyses for X-linked Adrenoleukodystrophy at the Biochemical Genetics Laboratory of the Institute of Human Genetics-National Institutes of Health, Manila

...

Machine Learning for the Biochemical Genetics Laboratory.

Machine Learning for the Biochemical Genetics Laboratory.

Newborn screening for CF in France: An exemplary national experience

Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France since 2002, with a 3-tiered strategy consisting in an immunoreactive trypsinogen (IRT) measurement at day-3, a search for the most common mutations responsible for CF when the IRT value is above the cut-off level, and, if necessary, a safetynet retesting of IRT at day-21. Coordination and follow-up are ensured at the national level and NBS is carried out through a regional organization involving NBS centers, biochemical and molecular genetics laboratories. Sweat testing and comprehensive mutation gene analysis are then performed according to a defined algorithm. Between 2002 and 2014, screening for the 30 most common mutations identified 87% of the alleles and comprehensive mutation gene analysis performed when applicable identified more than 300 additional mutations and resulted in a detection rate of 99.8% of the mutated alleles. Program surveillance ensured at a national level allowed to carry out adaptation of cut-off levels and removal of the p.Arg117His mutation. Thanks to these modifications, the performance of the French NBS program for CF meets the European guideline standards regarding positive predictive values, sensitivity and time to initial visit at the CF center, thus making the strategy effective. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

UPLC–MS/MS analysis of keratan sulfate from urine samples collected on filter paper for monitoring & follow-up of Morquio A patients

Since 2014, enzyme replacement therapy (ERT) has been available for treatment of Morquio A syndrome. During clinical trials, urinary keratan sulfate (KS) has been a useful biomarker and showed a marked decrease in patients on ERT, demonstrating therapy efficacy. Unfortunately, quantitative urinary KS testing is not widely available in biochemical genetics laboratories for efficient monitoring and follow-up of treated patients. A tandem mass spectrometry methodology was devised to analyze KS disaccharides and creatinine in urine specimens collected on filter paper. All Morquio A patients presented abnormal results pretreatment compared with reference values. This collection procedure can be performed by patients at home and filter papers sent by regular mail to a specialized laboratory, facilitating follow-up of patients.

Secondary Abnormal CSF Neurotransmitter Metabolite Profiles in a Pediatric Tertiary Care Centre.

Secondary neurotransmitter deficiencies have been reported in several reviews. Our primary aim was to assess the relationship among epilepsy, antiseizure medications, and specific neurotransmitter abnormalities. We also evaluated movement disorders and brain abnormalities via magnetic resonance imaging scans in patients with secondary neurotransmitter defects. This is a retrospective case series of 376 patients who underwent neurotransmitter analysis at BC Children's Hospital between 2009 and 2013, for a variety of neurological presentations. The biochemical genetics laboratory database was interrogated for results of cerebrospinal fluid neurotransmitter analyses. Clinical data for patients with abnormal results were collected from the hospital charts. Statistical analysis included one-way analysis of variance, chi-square, and a two-way contingency table. Abnormal neurotransmitter values were identified in 67 (17.8%) patients, two (0.53%) of which were attributable to a congenital neurotransmitter disorder and 11 (16.9%) secondary to other genetic diagnoses. Of 64 patients with secondary abnormal neurotransmitter values, 38 (59%) presented with epilepsy and 20 (31%) with movement disorders. A combination of epilepsy and movement disorder was less frequent. Acknowledging the limitations of this retrospective chart review, we conclude that, in our cohort, in addition to patients with movement disorders, a considerable number of patients with epilepsy and epileptic encephalopathy also showed secondary neurotransmitter mono-amine abnormalities. There is no clear relation, however, between clinical phenotype and type of neurotransmitter affected. In addition, no association was identified between the type of antiseizure medications and affected neurotransmitter type. We outline the need for prospective studies to further enrich our understanding of the relation between epilepsy and neurotransmitters with a focus on improving treatments and patient outcomes.

Allelic spectrum of formiminotransferase-cyclodeaminase gene variants in individuals with formiminoglutamic aciduria.

BackgroundElevated plasma and urine formiminoglutamic acid (FIGLU) levels are commonly indicative of formiminoglutamic aciduria (OMIM #229100), a poorly understood autosomal recessive disorder of histidine and folate metabolism, resulting from formiminotransferase‐cyclodeaminase (FTCD) deficiency, a bifunctional enzyme encoded by FTCD.MethodsIn order to further understanding about the molecular alterations that contribute to FIGLU‐uria, we sequenced FTCD in 20 individuals with putative FTCD deficiency and varying laboratory findings, including increased FIGLU excretion.ResultsIndividuals tested had biallelic loss‐of‐function variants in protein‐coding regions of FTCD. The FTCD allelic spectrum comprised of 12 distinct variants including 5 missense alterations that replace conserved amino acid residues (c.223A>C, c.266A>G, c.319T>C, c.430G>A, c.514G>T), an in‐frame deletion (c.1373_1375delTGG), with the remaining alterations predicted to affect mRNA processing/stability. These included two frameshift variants (c.990dup, c.1366dup) and four nonsense variants (c.337C>T, c.451A>T, c.763C>T, c.1607T>A).ConclusionWe observed additional FTCD alleles leading to urinary FIGLU elevations, and thus, providing molecular evidence of FTCD deficiency in cases identified by newborn screening or clinical biochemical genetic laboratory testing.

CAP/ACMG proficiency testing for biochemical genetics laboratories: a summary of performance

PurposeTesting for inborn errors of metabolism is performed by clinical laboratories worldwide, each utilizing laboratory-developed procedures. We sought to summarize performance in the College of American Pathologists’ (CAP) proficiency testing (PT) program and identify opportunities for improving laboratory quality. When evaluating PT data, we focused on a subset of laboratories that have participated in at least one survey since 2010.MethodsAn analysis of laboratory performance (2004 to 2014) on the Biochemical Genetics PT Surveys, a program administered by CAP and the American College of Medical Genetics and Genomics. Analytical and interpretive performance was evaluated for four tests: amino acids, organic acids, acylcarnitines, and mucopolysaccharides.ResultsSince 2010, 150 laboratories have participated in at least one of four PT surveys. Analytic sensitivities ranged from 88.2 to 93.4%, while clinical sensitivities ranged from 82.4 to 91.0%. Performance was higher for US participants and for more recent challenges. Performance was lower for challenges with subtle findings or complex analytical patterns.ConclusionUS clinical biochemical genetics laboratory proficiency is satisfactory, with a minority of laboratories accounting for the majority of errors. Our findings underscore the complex nature of clinical biochemical genetics testing and highlight the necessity of continuous quality management.

UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r2 > 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.

An Interview with David Millington.

David Millington was born under a lucky star. To hear him tell it, his life story consists of one serendipitous event after another. Certainly from a historical point of view, he has been at the right place at the right time. In 1941, just three years before he was born, the Americans George Beadle and Edward Tatum made a discovery that paved the way for the modern science of molecular biology. Mutating genes in Neurospora , a type of red bread mold, they were able to alter specific enzymes in the metabolic pathways of the mold, thereby confirming a hypothesis put forth in 1902 by the British physician Archibald Garrod that hereditary diseases are due to “inborn errors of metabolism.” This discovery was followed 20 years later—just as Millington was entering college—when the American microbiologist Robert Guthrie came up with the first blood test for phenylketonuria (PKU), which opened up the field of newborn screening. Millington wrote his own chapter in the tale in the late 1980s when he and colleagues had the idea to apply the cutting-edge technique of tandem mass spectrometry to the analysis of newborn blood samples. Their work—which has saved thousands of lives not just in North America but worldwide—revolutionized the field of newborn screening, turning it from a backwater into a scientific success story. In 2011, the Centers for Disease Control called newborn screening one of the great public health achievements of the past 10 years. Millington, who held the post of Medical Research Professor of Pediatrics and Director of the Biochemical Genetics Laboratory at Duke University Medical Center, spoke with me from his office in early July. He had just retired after over 30 years of service. This is a great moment—you've just retired! Two days ago. After a long and illustrious career. I …