M. Downing

Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency

SummaryGeneral mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5±0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0±0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of α- and β-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.

Carnitine-acylcarnitine translocase deficiency - a mild phenotype

A 15-month-old Pakistani child was admitted to hospital following a prolonged chronic convulsion. He had a depressed level of consciousness but physical examination was otherwise unremarkable. Plasma glucose was 1mmol/L and his urine contained no ketones. His condition improved rapidly with intravenous glucose. His parents are first cousins and his two older siblings are healthy. He had suffered two previous prolonged tonic febrile fits, during which his blood glucose had not been measured. On admission, his plasma lactate was 1.7mmol/L. Further investigations showed a plasma total carnitine of 13μmol/L (normal 23–60) and free carnitine of 4μmol/L (normal 15–53). Urine acylcarnitine was high (60μmol/mmol creatinine; normal 1.0–14.0) and urine free carnitine was low at <1.0μmol/mmol creatinine (normal 4.0–29.0). Serum insulin was appropriately low (0.6mU/L). Urine organic acids showed a hypoketotic dicarboxylic aciduria with prominent excretion of adipate and suberate. The sebacate to adipate ratio was <0.2. A small peak of suberylglycine was detected. He was put on a highcarbohydrate, low-fat diet with frequent feeds and L-carnitine 100mg/kg body weight per day, with the recommendation that he should not fast for more than 8h, subsequently amended to 6h. Dietary long-chain fat was set at a limit of 10g/day (estimation of previous long-chain fat intake was 40g/day) with medium-chain triglyceride supplementation and frequent high-carbohydrate feeds. He is now 20 years old and apart from two brief admissions to hospital during intercurrent infections, the most recent following a hypoglycaemic fit, his growth and development are normal. His cardiac function, as measured by ultrasonography, has remained normal throughout. Measurement of β-oxidation rates by the method of Manning and colleagues (1990) gave mean residual activities, as compared to simultaneous controls for [9,10-3H]myristate, [9,10-3H]palmitate and [9,10-3H]oleate, of 19%, 15%, 15% and 18%, 17% 14% for fibroblasts and lymphocytes, respectively. Measurement of carnitine-acylcarnitine translocase (McKusick 212138) activity by the method of Pande and colleagues (1993) gave 0.10 ±0.038 (8 repeats) vs 1.74±0.46 (n=9) nmol/min per kg protein for patient and controls, respectively. There have been six previous reports of carnitine-acylcarnitine translocase deficiency. All of these cases have had a severe phenotype with generally undetectable enzyme activity and very low β-oxidation flux. All of these patient have had a fatal outcome, most in the neonatal period, although one child survived to 3 years with early medical intervention. Our patient gave a mean residual carnitine-acylcarnitine translocase activity of 6% of controls and a mean residual β-oxidation activity in lymphocytes and fibroblasts of 16%. J. Inher. Metab. Dis. 20 (1997) 000–000

Implementation of the newborn screening programme for sickle cell disease in England: results for 2003-2005.

Objectives This paper reports early screening results from the newborn sickle cell disease screening programme recently implemented in England. Setting England. Screening is offered at 5-8 days of age as part of the existing bloodspot test and offered to all babies irrespective of ethnicity. Methods The laboratory methods recommended are high performance liquid chromatography (HPLC) and iso-electric focusing (IEF). 15 Two methods of analysis must be applied to all screen positive results. The conditions screened for are:- Sickle cell anaemia (Hb SS), Hb SC disease, Hb S/β-thalassaemia, Hb S/DPunjab, Hb S/OArab, Hb S/HPFH. Carriers identified for the common haemoglobin variants are reported to parents and follow-up counselling is offered. A bespoke laboratory quality assurance programme has been established which has defined standards of satifactory performance. Results Provisional figures from the first seven months of screening (up to March 2004) 108,255 infants were screened gave a screen positive rate of 1:900 for these high prevalence areas and a carrier rate of 2.7%. Figures for 2004-2005 show about 250 significant screen positive results for sickle cell disorders and about 6,500 carriers were identified. The birth prevalence for screen positive results from 2004-05 is 1:1500. We estimate that when there is countrywide data, the national birth prevalence will be about 1:2000-1:2,500. Conclusion The results from the national newborn sickle cell screening programme in England - show that the sickle cell disorders are as common as cystic fibrosis (CF) in England, although the distribution of cases is concentrated in London and other urban areas. The findings and approach to implementation adopted in England may be of interest to other Western European countries with increasing rates of sickle cell disease who are considering such programmes and also to other developed countries.

Newborn screening for medium chain acyl-CoA dehydrogenase deficiency in England: prevalence, predictive value and test validity based on 1.5 million screened babies

Background Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a rare, life-threatening condition. Early diagnosis by screening asymptomatic newborns may improve outcome, but the benefit to newborns identified with variants not encountered clinically is uncertain. Objective To estimate, overall and by ethnic group: screen-positive prevalence and predictive value (PPV); MCADD prevalence; proportion MCADD variants detected of predicted definite or uncertain clinical importance. Setting All births in areas of high ethnic minority prevalence in England. Methods Prospective multicentre pilot screening service; testing at age five to eight days; standardized screening, diagnostic and management protocols; independent expert review of screen-positive cases to assign MCADD diagnosis and predicted clinical importance (definite or uncertain). Results Approximately 1.5 million babies (79% white; 10% Asian) were screened. MCADD was confirmed in 147 of 190 babies with a positive screening result (screen-positive prevalence: 1.20 per 10,000; MCADD prevalence: 0.94 per 10,000; PPV 77% [95% CI 71–83]), comprising 103 (70%) with MCADD variants of definite clinical importance (95 white [95%]; 2 Asian [2%]) and 44 (30%) with variants of uncertain clinical importance (29 white [67%]; 12 Asian [28%]). Conclusion One baby in every 10,000 born in England is diagnosed with MCADD by newborn screening; around 60 babies each year. While the majority of MCADD variants detected are predicted to be of definite clinical importance, this varies according to ethnic group, with variants of uncertain importance most commonly found in Asian babies. These findings provide support for MCADD screening but highlight the need to take account of the ethnic diversity of the population tested at implementation.

Ethnicity of children with homozygous c.985A>G medium-chain acyl-CoA dehydrogenase deficiency: findings from screening approximately 1.1 million newborn infants.

Objectives It has been suggested that homozygous c.985A>G medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disease of White ethnic origin but little is known regarding its ethnic distribution. We estimated ethnic-specific homozygous c.985A>G MCADD birth prevalence from a large-scale UK newborn screening study. Methods Homozygous c.985A>G MCADD cases were ascertained in six English newborn screening centres between 1 March 2004 and 28 February 2007 by screening approximately 1.1 million newborns using tandem mass spectrometry analysis of underivatised blood spot samples to quantitate octanoylcarnitine (C8). Follow-up biochemistry and mutation analyses for cases (mean triplicate C8 value ≥0.5 µmol/L) were reviewed to confirm diagnosis. Ethnicity was ascertained from clinician report and denominators from 2001 UK Census estimates of ethnic group of children less than one year. Results Sixty-four infants were c.985A>G MCADD homozygotes (overall prevalence 5.8 per 100,000 live births; 95% CI 4.4–7.2). Sixty (93%) were White, two (3%) were mixed/other and two were of unknown ethnic origin. No Asian or Black homozygotes were identified. Proportions of White, mixed/other, Asian and Black births in screening regions were estimated, yielding homozygous c.985A>G MCADD birth prevalence of 6.9 per 100,000 (95% CI 5.2–8.8) in White, and 95% CI estimates of 0–2.7 per 100,000 in Asian and 0–5.8 in Black populations. The c.985A>G carrier frequency in the White group was estimated at one in 65 (95% CI 1/74, 1/61) under Hardy–Weinberg conditions. Conclusion c.985A>G homozygous MCADD is not found in Black and Asian ethnic groups that have been screened at birth in England. This is consistent with the earlier published observations suggesting that MCADD due to the c.985A>G mutation is a disease of White ethnic origin.

Detection of urinary hexanoylglycine in the diagnosis of MCAD deficiency from newborn screening.

We strongly support Piero Rinaldo_s and Kevin Carpenter_s comments regarding the importance of accurate and reliable identification of urinary hexanoylglycine (HG) as a definitive diagnostic factor for MCAD deficiency (J Inherit Metab Dis 2008;31: 142–145, Notes and Queries in Metab-L). Qualitative organic acid analysis should detect the near normal concentrations of HG seen in some Fnon-crisis_ MCADD (both c.985A>G homozygotes and mild variants) samples. However, data from the ERNDIM EQA scheme with >70 participants demonstrate failure to detect HG around 8 2mol/mmol creatinine in 10–30% of samples. When the UK embarked on pilot screening for MCADD in 2004 the six UKCSNS-MCADD screening centres (Shortland et al 2006) agreed that, after qualitative organic acid assessment of trimethylsilyl derivatives by individual laboratories, all follow-up urine samples from presumptive positive cases (day 5 blood spot octanoylcarnitine >0.5 2mol/L) would have HG quantified by a central laboratory using GC-MS stable-isotope dilution analysis (SID) of methyl esters. To our knowledge, quantitative HG data have not been available in other studies of newborn screening for MCADD. In the two-year pilot period, HG concentrations (2mol/mmol creatinine) were available in 93/105 cases: 6 but in only 1/13 samples below the upper limit of normal (<1.1). Detection of HG failed in 4 samples with concentrations above the normal range (values 1.2, 5.0, 5.5 and 5.9). In view of these findings, for implementation of MCADD screening in the UK (completion due April 2009) the UKCSNS-MCADD recommended that follow up urine samples where HG is not detected on qualitative analysis should have HG quantified by GCMS-SID.

Fumarate hydratase deficiency: increased fumaric acid in amniotic fluid of two affected pregnancies

Bakker HD, Van Den Bogert C, Scholte HR et al (1996) Fatal neonatal liver failure and depletion of mitochondrial DNA in three children of one family. J Inherit Metab Dis 19: 112^115. Mazziotta MRM, Ricci E, Bertini E, et al (1992) Fatal infantile liver failure associated with mitochondrial DNA depletion. J Pediatr 121: 896^901. Moraes CT, Schanske S, Tritschler HJ, et al (1991) mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am J Hum Genet 48: 492^501. Poulton J, Morten K, Freeman-Emmerson C, et al (1994) De¢ciency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum Mol Genet 3: 1763^1769. Vu TH, Sciacco M, Tanji K, et al (1998) Clinical manifestation of mitochondrial DNA depletion. Neurology 50: 1783^1790.

Problems in the detection of fatty acid oxidation defects: Experience of a quality assurance programme for qualitative urinary organic acid analysis

An external quality assurance scheme for qualitative urinary organic acid analysis was started in 1992 with 23 participants, mostly from the United Kingdom (Bonham et al 1994), and has steadily grown under the ERNDIM (European Research Network for Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Metabolic Disorders) initiative to the current 111 participants from 28 countries worldwide. Eighty-nine per cent of participants use GC-MS, 10% use GC only and there is one HPLC user. Ether and/or ethyl acetate extraction with trimethylsilyl derivatization is used by > 90% of laboratories. Twelve samples are circulated annually. Each sample comprises 2 ml heat-treated urine (not spiked but sometimes pooled) from children who have a known disorder or who are normal. Brief clinical details are supplied and participants are asked to report in the usual way used in that laboratory and to include an annotated chromatogram. Urine samples from patients with 26 different disorders have been circulated to date and there has been an improvement in the rate of recognition of some of these conditions, supporting the educational value of this scheme. However, the identification of fatty acid oxidation defects is a continuing problem for some laboratories, from both an analytical and an interpretational standpoint.

Normal acylcarnitines in maternal urine during a pregnancy affected by glutaric aciduria type II

Normal acylcarnitines in maternal urine during a pregnancy a†ected by glutaric aciduria type II N. J. Manning1*, J. R. Bonham1, M. Downing1, R. G. Edwards1, S. E. Olpin1, R. J. Pollitt1, M. Pourfarzam3, M. J. Sharrard2 and M. S. T anner2 1 Department of Chemical Pathology and Neonatal Screening, 2 Department of Paediatrics, The ChildrenÏs Hospital, Sheffield ; 3 Department of Child Health, The Royal Victoria InÐrmary, Newcastle upon Tyne, UK * Correspondence : Department of Chemical Pathology and Neonatal Screening, The ChildrenÏs Hospital, Western Bank, Sheffield S10 2TH, UK