Metabolic Serendipities of Expanded Newborn Screening.

Incidental findings on newborn screening (NBS) are results that are not the target of screening within a given NBS program, but rather are found as a result of the screening and resulting diagnostic workup for that target. These findings may not have an immediate clinical impact on the newborn, but are sometimes an additional benefit of NBS programs and may be considered secondary targets of NBS programs. This work describes four case reports that had incidental findings on the NBS, which eventually led to the diagnosis of another metabolic disease instead of the one that was initially suspected. The first case was a new defect in the cationic amino acid transporter-2 (CAT-2), which was oriented as an arginase-1 deficiency in the newborn. The second case was a maternal glutaric aciduria type 1 (GA-1) that mimicked a carnitine transporter deficiency in the newborn. The third report was a case of lysinuric protein intolerance (LPI), which appeared as high levels of citrulline on the NBS. The fourth case was a mother with homocystinuria that was diagnosed during the biochemical study of vitamin B12 status. All cases provide new or interesting data that will help guide differential diagnosis in the future.

Ethnic variability in newborn metabolic screening markers associated with false-positive outcomes.

Newborn screening (NBS) programmes utilise information on a variety of clinical variables such as gestational age, sex, and birth weight to reduce false-positive screens for inborn metabolic disorders. Here we study the influence of ethnicity on metabolic marker levels in a diverse newborn population. NBS data from screen-negative singleton babies (n = 100 000) were analysed, which included blood metabolic markers measured by tandem mass spectrometry and ethnicity status reported by the parents. Metabolic marker levels were compared between major ethnic groups (Asian, Black, Hispanic, White) using effect size analysis, which controlled for group size differences and influence from clinical variables. Marker level differences found between ethnic groups were correlated to NBS data from 2532 false-positive cases for four metabolic diseases: glutaric acidemia type 1 (GA-1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). In the result, 79% of the metabolic markers (34 of 43) had ethnicity-related differences. Compared to the other groups, Black infants had elevated GA-1 markers (C5DC, Cohen's d = .37, P < .001), Hispanics had elevated MMA markers (C3, Cohen's d = .13, P < .001, and C3/C2, Cohen's d = .27, P < .001); and Whites had elevated VLCADD markers (C14, Cohen's d = .28, P < .001, and C14:1, Cohen's d = .22, P < .001) and decreased OTCD markers (citrulline, Cohen's d = -.26, P < .001). These findings correlated with the higher false-positive rates in Black infants for GA-1, in Hispanics for MMA, and in Whites for OTCD and for VLCADD. Web-based tools are available to analyse ethnicity-related changes in newborn metabolism and to support developing methods to identify false-positives in metabolic screening.

Elevated levels of BDNF and cathepsin-d as possible peripheral markers of neurodegeneration in plasma of patients with glutaric acidemia type I.

Glutaric acidemia type I (GA1) is caused by severe deficiency of glutaryl-CoA dehydrogenase activity, resulting in an accumulation of glutaric acid and glutarylcarnitine (C5DC) in the organism. Patients affected by GA1 are asymptomatic in the neonate period but usually manifest chronically progressive neurodegeneration apart from severe encephalopathic crises associated with acute striatum necrosis. Neurological manifestations like dyskinesia, dystonia, hypotonia, muscle stiffness, and spasticity are present. Treatment is based on protein/lysine restriction and l-carnitine supplementation. In this work, we evaluated markers of neurodegeneration and inflammation, namely BDNF (brain-derived neurotrophic factor), NCAM (neuronal adhesion molecule), PDGF-AA (platelet-derived growth factor), and cathepsin-d in plasma of six treated GA1 patients. We first found marked increases of plasma C5DC concentrations in GA1 patients, as well as increased levels of the markers BDNF and cathepsin-d as compared to those of age-matched healthy children. Furthermore, C5DC concentrations were highly correlated with the levels of cathepsin-d. These results may demonstrate that brain tissue degeneration is present in GA1 patients and that there is a relationship between increased metabolites concentrations with this process. To the best of our knowledge, this is so far the first study showing altered peripheral parameters of neurodegeneration and inflammation in GA1 patients.

Melatonin improves hyperglycemia induced damages in rat brain.

BACKGROUND: Diabetes mellitus is an endocrine disorder which is characterized by the development of resistance to the cellular activity of insulin or inadequate insulin production. It leads to hyperglycemia, prolonged inflammation, and oxidative stress. Oxidative stress is assumed to play an important role in the development of diabetic complications. Melatonin is the hormone that interacts with insulin in diabetes. Therefore, in this study, the effects of melatonin treatment with or without insulin were examined in diabetic rat brain. METHODS: Rats were divided into five groups as control, diabetes, diabetes + insulin, diabetes + melatonin, and diabetes + melatonin + insulin. Experimental diabetes was induced by streptozotocin (60 mg/kg, i.p.). Twelve weeks after diabetes induction, rats were decapitated. Malondialdehyde, glutathione, sialic acid and nitric oxide levels, superoxide dismutase, catalase, glutathione-S-transferase, myeloperoxidase, and tissue factor activities were determined in brain tissue. RESULTS: Melatonin alone showed its antioxidant effect by increasing brain glutathione level, superoxide dismutase, catalase, and glutathione-S-transferase activities and decreasing malondialdehyde level in experimental diabetes. Although insulin did not have a significant effect on glutathione and glutathione-S-transferase, its effects on lipid peroxidation, superoxide dismutase, and catalase were similar to melatonin; insulin also decreased myolopeoxidase activity and increased tissue factor activity. Combined melatonin and insulin treatment mimicked the effects of insulin. CONCLUSION: Addition of melatonin to the insulin treatment did not change the effects of insulin, but the detailed role of melatonin alone in the treatment of diabetes merits further experimental and clinical investigation.

Selective Screening of Fatty Acids Oxidation Defects and Organic Acidemias by Liquid Chromatography/tandem Mass Spectrometry Acylcarnitine Analysis in Brazilian Patients.

BACKGROUND: Inborn errors of metabolism (IEM) are diseases which can lead to accumulation of toxic metabolites in the organism. AIM OF THE STUDY: To investigate, by selective screening, mitochondrial fatty acid oxidation defects (FAOD) and organic acidemias in Brazilian individuals with clinical suspicion of IEM. METHODS: A total of 7,268 individuals, from different regions of Brazil, had whole blood samples impregnated on filter paper which were submitted to the acylcarnitines analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre, Brazil, during July 2008-July 2016. RESULTS: Our results showed that 68 patients (0.93%) were diagnosed with FAOD (19 cases) and organic acidemias (49 cases). The most prevalent FAOD was multiple acyl CoA dehydrogenase deficiency (MADD), whereas glutaric type I and 3-OH-3-methylglutaric acidemias were the most frequent disorders of organic acid metabolism. Neurologic symptoms and metabolic acidosis were the most common clinical and laboratory features, whereas the average age of the patients at diagnosis was 2.3 years. CONCLUSIONS: Results demonstrated a high incidence of glutaric acidemia type I and 3-OH-3- methylglutaric acidemia in Brazil and an unexpectedly low incidence of FAOD, particularly medium-chain acyl-CoA dehydrogenase deficiency (MCADD).

Favourable outcome in a child with symptomatic diagnosis of Glutaric aciduria type 1 despite vertical HIV infection and minor head trauma.

The first case of Glutaric aciduria Type 1(GA1) in an African child was reported in 2001. GA1 has a prevalence of 1:5000 in black South Africans. Although early diagnosis is essential for a favourable outcome, newborn screening is not routine in South Africa where an estimated 320,000 children have HIV infection. Neurodevelopmental delay and encephalopathy are complications of both HIV and GA1. In such a setting it is important to recognise that HIV and GA1 can occur simultaneously. We present an HIV-infected South African male child of Xhosa descent with macrocephaly who commenced combination antiretroviral therapy (ART) at 8 weeks of age in a clinical trial which included a neurodevelopmental sub-study. He developed short-lived focal seizures at 16 months after minor head trauma. Neurological examination was normal. Neuroimaging showed temporal lobe atrophy, subtle hyperintense signal change in the globus pallidus, and focal haemosiderosis in the right Sylvian fissure region. As findings were not in keeping with HIV encephalopathy, a urine metabolic screen was undertaken which suggested GA1. Genetic testing confirmed Arg293Trp mutation. He began L-carnitine and a low protein diet as a restricted diet was not practicable. At 21 months he developed pulmonary tuberculosis, requiring 6 months treatment. He did not develop any neurologic motor symptoms. Serial neurodevelopmental and neuropsychological test scores until 9 years were similar to healthy neighbourhood controls, except for mild language delay at 3½ years. Detection of GA1, probably facilitated through participation in a clinical trial, was pivotal for a favourable outcome. The concomitant use of ART and anti-tuberculous therapy in a child with GA1 appears safe.

Recurrent rhabdomyolysis and glutaric aciduria type I: a case report and literature review.

BACKGROUND: Glutaric acidemia type I (GA-I) is a rare metabolic disorder caused by mutation of the glutaryl- CoA dehydrogenase (GCDH) gene. The occurrence of rhabdomyolysis with GA-I is extremely rare. METHODS: We reported a child with recurrent rhabdomyolysis and undiagnosed glutaric acidemia type I (GA-I). And a literature review was performed. RESULTS: A 4.5-year-old girl was admitted to our hospital due to recurrent rhabdomyolysis for 3 times within three years. At the third admission, she was diagnosed with GA-I by biochemical testing and mutation analysis. The girl was found to have a serine to leucine replacement mutation of the GCDH gene in exon 8 at position 764. Other three patients with rhabdomyolysis and GA-I were discovered by literature searching. CONCLUSIONS: This report highlights that patients with GA-I may have an increased risk of rhabdomyolysis.

Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots.

BACKGROUND: Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. METHODS: For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. RESULTS: The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. CONCLUSION: The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies.

Adult-onset glutaric aciduria type I presenting with white matter abnormalities and subependymal nodules.

A 55-year-old female presented with a 6-year history of paresthesias, incontinence, spasticity, and gait abnormalities. Neuroimaging revealed white matter abnormalities associated with subependymal nodules. Biochemical evaluation noted increased serum C5-DC glutarylcarnitines and urine glutaric and 3-hydroxyglutaric acids. Evaluation of the glutaryl-CoA dehydrogenase (GCDH) gene revealed compound heterozygosity consisting of a novel variant (c.1219C>G; p.Leu407Val) and pathogenic mutation (c.848delT; p.L283fs). Together, these results were consistent with a diagnosis of adult-onset type I glutaric aciduria.

Treating Every Needle in the Haystack: Hyperammonemic Encephalopathy and Severe Malnutrition After Bariatric Surgery-A Case Report and Review of the Literature.

Neurologic complications are not uncommon following bariatric surgery. Hyperammonemic encephalopathy (HAE) due to an acquired or unmasked urea cycle deficit is among the rarest of these. Pediatric nutrition support specialists are familiar with recognizing urea cycle deficits, but adult specialists may not be. Here we present a case of a patient initially misdiagnosed with cirrhosis who presented with recurrent HAE 4 years after Roux-en-Y gastric bypass. She was diagnosed with a proximal urea cycle deficit and severe protein calorie malnutrition. The patient recovered with specialized nutrition and medical support targeting this condition. A literature review indicates multiple fatalities from this condition, indicating the importance of early diagnosis and appropriate nutrition support.

Neuroimaging in patients with abnormal blood glucose levels.

SUMMARY: Smooth neuronal functioning requires an uninterrupted supply of energy that is provided by glucose under normal physiologic conditions. Significant variations in plasma glucose levels, be it hypoglycemia or hyperglycemia, can present with myriad clinical manifestations and may mimic stroke. At times, the diagnosis is either not apparent or not clinically suspected. Imaging can suggest the diagnosis in unsuspected cases and can help in the assessment of the extent of neuronal damage in known cases, making it vital for the neuroradiologist to be aware of both common and atypical neuroimaging findings in hypoglycemia and hyperglycemia.

Hyponatremia following mild/moderate subarachnoid hemorrhage is due to SIAD and glucocorticoid deficiency and not cerebral salt wasting.

CONTEXT: Hyponatremia is common after acute subarachnoid hemorrhage (SAH) but the etiology is unclear and there is a paucity of prospective data in the field. The cause of hyponatremia is variously attributed to the syndrome of inappropriate antidiuresis (SIAD), acute glucocorticoid insufficiency, and the cerebral salt wasting syndrome (CSWS). OBJECTIVE: The objective was to prospectively determine the etiology of hyponatremia after SAH using sequential clinical examination and biochemical measurement of plasma cortisol, arginine vasopressin (AVP), and brain natriuretic peptide (BNP). DESIGN: This was a prospective cohort study. SETTING: The setting was the National Neurosurgery Centre in a tertiary referral centre in Dublin, Ireland. PATIENTS: One hundred patients with acute nontraumatic aneurysmal SAH were recruited on presentation. INTERVENTIONS: Clinical examination and basic biochemical evaluation were performed daily. Plasma cortisol at 0900 hours, AVP, and BNP concentrations were measured on days 1, 2, 3, 4, 6, 8, 10, and 12 following SAH. Those with 0900 hours plasma cortisol<300 nmol/L were empirically treated with iv hydrocortisone. MAIN OUTCOME MEASURES: Plasma sodium concentration was recorded daily along with a variety of clinical and biochemical criteria. The cause of hyponatremia was determined clinically. Later measurement of plasma AVP and BNP concentrations enabled a firm biochemical diagnosis of the cause of hyponatremia to be made. RESULTS: Forty-nine of 100 developed hyponatremia<135 mmol/L, including 14/100<130 mmol/L. The cause of hyponatremia, and determined by both clinical examination and biochemical hormone measurement, was SIAD in 36/49 (71.4%), acute glucocorticoid insufficiency in 4/49 (8.2%), incorrect iv fluids in 5/49 (10.2%), and hypovolemia in 5/49 (10.2%). There were no cases of CSWS. CONCLUSIONS: The most common cause of hyponatremia after acute nontraumatic aneurysmal SAH is SIAD. Acute glucocorticoid insufficiency accounts for a small but significant number of cases. We found no cases of CSWS.

Glutaric aciduria type I: outcome of patients with early- versus late-diagnosis.

Patients with Glutaric aciduria type 1 (GA-1) can be identified by newborn screening using tandem mass spectrometry. The clinical evolution of screened patients seems to be more favourable compared with those diagnosed later, although long-term evolution is still doubtful. We have evaluated the outcome in nine GA-1 patients diagnosed in our region during 12 years. Six were detected by newborn screening and 3 clinically. The birth prevalence was 1:35,027. High blood C5DC concentration, in 8/9 patients, was found, whereas all patients exhibited high concentration of this metabolite in urine. Therefore, urine C5DC was a good marker for the detection of this disease. Eight different mutations in the GCDH gene were identified, four of them were novel (p.R88H, p.Y398C, p.R372K, p.D220N); being p.R227P the mostcommon. Macrocephaly with enlarged frontotemporal subarachnoid space was present in 4/6 patients diagnosed by newborn screening, all these patients required high energy intake, and in two cases, enteral feeding during the first year of life was needed. One child had an intercurrent episode of feeding refuse with hypoglycemia at two years of age. The mean follow-up time of screened patients was 56 months, and patients still remain asymptomatic. However, after a mean follow-up of 97 months treatment efficacy was poor in unscreened patients, two of them showing a severe spastic tetraparesis. Plasma levels of lysine, tryptophan and carnitine, were the most useful biomarkers for the follow-up. Our data support that, early diagnosis and treatment strategies are essential measures for the good clinical evolution of GA-1 patients.

Low lysine diet in glutaric aciduria type I--effect on anthropometric and biochemical follow-up parameters.

BACKGROUND: Metabolic treatment in glutaric aciduria type I (GA-I) including a low lysine diet with lysine-free, tryptophan-reduced amino acid supplements (AAS), carnitine supplementation and early start of emergency treatment during putatively threatening episodes of intermittent febrile illness dramatically improves the outcome and thus has been recommended by an international guideline group (Kölker et al, J Inherit Metab Dis 30:5-22, 2007). However, possible affection of linear growth, weight gain and biochemical follow-up monitoring has not been studied systematically. METHODS: Thirty-three patients (n = 29 asymptomatic, n = 4 dystonic) with GA-I who have been identified by newborn screening in Germany from 1999 to 2009 were followed prospectively during the first six years of life. Dietary treatment protocols, anthropometrical and biochemical parameters were longitudinally evaluated. RESULTS: Mean daily intake as percentage of guideline recommendations was excellent for lysine (asymptomatic patients: 101 %; dystonic patients: 103 %), lysine-free, tryptophan-reduced AAS (108 %; 104 %), energy (106 %; 110 %), and carnitine (92 %; 102 %). Low lysine diet did not affect weight gain (mean SDS 0.05) but mildly impaired linear growth in asymptomatic patients (mean SDS -0.38), while dystonic patients showed significantly reduced weight gain (mean SDS -1.32) and a tendency towards linear growth retardation (mean SDS -1.03). Patients treated in accordance with recent recommendations did not show relevant abnormalities of routine biochemical follow-up parameters. INTERPRETATION: Low lysine diet promotes sufficient intake of essential nutrients and anthropometric development in asymptomatic children up to age 6 year, whereas individualized nutritional concepts are required for dystonic patients. Revised recommendations for biochemical monitoring might be required for asymptomatic patients.

Glutaric acidemia type 1: outcomes before and after expanded newborn screening.

Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism. Patients may present with brain atrophy, macrocephaly, and acute dystonia secondary to striatal degeneration typically triggered by an infection, fever, and/or dehydration. This disorder is identified on expanded newborn screening by increased glutarylcarnitine. We evaluated the outcome of 19 patients with GA-1. Ten patients were diagnosed by newborn screening and 9 were diagnosed clinically. DNA testing in 12 patients identified 15 different mutations in the glutaryl-CoA dehydrogenase gene. Plasma glutarylcarnitine and urinary 3-hydroxyglutaric acid were elevated in all patients. However, only 10 of 17 patients who underwent urine organic acid analysis were high excretors of glutaric acid. Levels of glutarylcarnitine in plasma correlated with the urinary excretion of glutaric and 3-hydroxyglutaric acid, but not with clinical outcome. Plasma lysine was also significantly correlated with urinary glutaric acid, but not with urinary 3-hydroxyglutaric acid. Brain magnetic resonance imaging in all patients showed wide Sylvian fissures before treatment, which normalized by 4 years of age in treated patients. The occurrence of three adverse outcomes (oral motor function, ambulatory capability, and dystonic movements) was on average reduced by 75% (relative risk 0.25 to 0.28) in patients identified by newborn screening compared to patients diagnosed before newborn screening (Fisher's exact test; p=0.0055 for oral motor function and ambulatory capability; p=0.023 for dystonic movements). Newborn screening is effective in the prevention of complications in patients with GA-1 when coupled with treatment strategies.

Avoid adding insult to injury - correct management of sick female endurance athletes.

OBJECTIVES: To evaluate the efficacy of Ringer's lactate, isotonic saline and hypertonic saline on the clinical and biochemical recovery of athletes with exercise-associated hyponatraemic encephalopathy caused by fluid overload. METHODS: We retrospectively reviewed serial blood sodium concentrations (Na+) and qualitative signs of recovery and time to recovery in two healthy menstruant females hospitalised with dilutional exercise-associated hyponatraemic encephalopathy after withdrawal from the 2011 Comrades Marathon (89 km) and Argus Cycle Tour (109 km). RESULTS: Improvements in blood Na+ did not occur with intravenous administration of Ringer's lactate solution, but did occur with administration of isotonic and hypertonic saline. Qualitative improvements in mental status were not quantitatively related to the biochemical value of blood Na+ or subsequent return to normonatraemia. CONCLUSIONS; Hyponatraemia should be suspected in all female athletes presenting to the medical area of endurance races with vomiting, altered mental status and a history of high fluid intake. If a diagnosis of exercise-associated hyponatraemia with cerebral encephalopathy is confirmed, the treatment of choice is administration of an intravenous bolus of hypertonic saline. Administration of Ringer's lactate should be discouraged, as this does not correct Na+ and appears to delay recovery.

Serum calcium concentration affects signal changes on diffusion-weighted imaging in hypoglycemic encephalopathy.

BACKGROUND AND PURPOSE: Abnormal signals in brain DWI may appear in patients with HE. The aim of this study was to compare the clinical condition and various physiologic factors between patients with HE with and without abnormal signal intensity changes on DWI. MATERIALS AND METHODS: We retrospectively enrolled patients with HE who underwent brain DWI studies from January 2002 to November 2010. A diagnosis of HE was defined as low serum glucose levels (<50 mg/dL) with alteration of consciousness. Several clinical conditions and physiologic parameters were compared between patients with and without abnormal signals on DWI, including consciousness levels; outcome; body temperature; blood pressure; and serum levels of glucose, calcium, sodium, blood urea nitrogen, and creatinine. RESULTS: Nine patients with HE were included, and 3 of them (33%) had abnormal signals on brain DWI. There was a trend toward serum calcium concentrations being lower in patients with normal findings on DWI studies compared with patients with abnormal DWI signals (7.6 ± 1.7 versus 9.4 ± 0.7 mg/dL, P = .07). Serum glucose concentration, duration of hypoglycemia, consciousness levels, other physiologic parameters, and clinical outcome did not reveal any differences between the 2 groups. CONCLUSIONS: One-third of patients with HE had abnormal signals on brain DWI, and patients with low serum calcium levels may be less likely to present with abnormal DWI signals.

An improved LC-MS/MS method for the detection of classic and low excretor glutaric acidemia type 1.

Glutaric acidemia type I (GA1) is associated with elevated glutarylcarnitine (C5DC), typically measured as its butylester by acylcarnitine profile analysis using tandem mass spectrometry (MS/MS) and the precursor-product ion pair of m/z 388-85. This method neither distinguishes between C5DC and its isomer 3-hydroxydecanoylcarnitine (C10-OH) nor reliably detects the low-excretor variant of GA1, leading to both false-positive and false-negative results when testing for GA1. To overcome these limitations, we developed an LC-MS/MS method that discriminates C5DC from C10-OH by the use of precursor-product ion pairs specific for butylated C5DC (m/z 388-115) and underivatized C10-OH (m/z 332-85). The C5DC method was validated over the linearity range of 0.025-20 µM with a lower limit of quantification (LOQ) of 0.025 µM. Excellent precision and accuracy were also observed. We tested plasma samples from 10 patients with confirmed GA1 (including 3 with the low-excretor variant), 21 patients with mild elevations of C5DC or C10-OH by routine acylcarnitine analysis for which GA1 ultimately was excluded, and 29 normal controls. By using the m/z 388-115 ion pair, all cases of GA1, including the low-excretor variant, were reliably distinguished from normal controls. By using the m/z 388-85 pair, patients with ambiguous elevations of C5DC or C10-OH demonstrated clearly elevated levels of C10-OH (m/z 332-85) but normal C5DC (m/z 388-115), confirming that the apparent elevation of C5DC is due to interference by C10-OH. Our method results in excellent detection of GA1, including the low-excretor variant, and also provides a means to discriminate C5DC and C10-OH in follow-up testing and routine acylcarnitine studies.