and distribution into the qPCR plates, the extracted DNA and controls DNA (comprising all possible genotypes) are loaded into the equipment and transferred their specific qPCR wells. After the thermocycling, the amplification data is transferred to the integrator software, inspected by the operator together with the melting curves and the approved results are sent to LIS without any typing. The workflow can process 82 samples total (irrespective of the requested test) in 5 h, have complete traceability, and for its validation, the obtained by results was compared with the expected results. Results The automated workflow attributed the expected genotypes for all samples in all instances: 223 GG, 13 GA and 3 AA for factor V Leiden; 144 GG, 10 GA and 0 AA for Prothrombin G20210A; 29 positive and 206 negative for HLA-B27; 28 CC, 38 CT and 7 TT for Lactase C − 13,910 T; MTHFR 43 CC, 46 CT, and 10 TT for MTHFR C677T and 48 GG, 3 GA, 1 AA for HFE C282Y and 35 CC, 14 CG, 3 GG HFE H63D mutation in hemochromatosis gene. Conclusions The proposed automated qPCR workflow could accurately genotype seven distinct SNP. Its full automation confers higher safety and quality for the process. doi:10.1016/j.cca.2019.03.1189 W271 Mitochondrial DNA depletion and clinical presentations M. Chtourou a , S. Schaeffer b , F. Chapon c,d , S. Allouche a a CHU de Caen, Department of Biochemistry, Caen F-14032, France b CHU de Caen, Neuromuscular Competence Center, Caen F-14032, France c Department of Pathology, Caen F-14032, France d CHU de Caen, Neuromuscular Competence Center, Caen F-14032, France Background-aim Human mtDNA (mitochondrial DNA), which encodes 13 subunits of the oxidative phosphorylation complexes, is under control of nuclear genes involved in nucleotide synthesis and DNA replication (Viscomi and Zeviani, 2017). Depletion, which is defined as a reduction of mtDNA copy number (b30% of the age-matched controls), is responsible for a heterogeneous group of autosomal recessive diseases with different clinical presentations (hepa- tocerebral, myopathic, encephalomyopathy and neu- rogastrointestinal) named mtDNA depletion syndromes (MDS). mtDNA depletion was also reported in patients with severe myopathy, proximal myopathy, spinal muscular atrophy and neuro- muscular diseases (Katsetos et al., 2013; Komulainen et al., 2015; Ripolone et al., 2015; Brady et al., 2016). The purpose of our study was to describe the clinical presenta- tions, the biochemical and histological abnormalities detected in the muscle biopsy in which the mtDNA copy number was decreased. Methods Quantification of mtDNA was performed in muscle biopsy from 117 patients by quantitative PCR (QPCR) from January 2017 to December 2018 with amplification of the mitochondrial 16S and the nuclear 18S rRNA. Results The mtDNA depletion was diagnosed in 27 patients but clinical, biochemical, histological and genetic information was available in 25 patients that were subdivided into group 1 (b18 years) and group 2 (N18 years). The group 1 included 7 patients presenting with seizure/ encephalopathy (4/7), suspicion of mitochondrial diseases (3/7) and myopathy (1/7). A respiratory chain deficiency was detected in (3/7) patients but no histological abnormality was evidenced. The second group included 18 patients presenting with myalgia (4/18), a proximal progressive muscle weakness (8/18), neuropathy (3/18) and increased serum CK level (9/18). Muscle biopsy showed inclusion body myositis (5/18), nuclear internalization (3/18), and marked fibers size variability (3/18) and a respiratory chain deficiency was detected in (3/18). High throughput sequencing of genes involved in mtDNA maintenance was performed in 4 patients from group 1 and 3 patients from group 2 but showed no pathogenic mutation. Conclusions In children, mtDNA depletion is associated with respiratory chain deficiency and proximal progressive muscle weakness, increased serum CK and inclusion body myositis in adult patients. doi:10.1016/j.cca.2019.03.1190 W272 Characterization PHA genotype and the BH4 responsive in HPA patients M. De Toro Crespo b , J. Liró Armenteros b , M. Bueno Delgado c , C. Delgado Pecellin a , J.M. Guerrero Montalvez a , H. Macher Manzano a a Clinical Biochemical Department, Hospital Universitario Virgen del Rocío, Sevilla, Spain b Clinical Biochemical Department, Hospital Universitario Virgen Mac- arena, Sevilla, Spain c Pediatric Department, Hospital Universitario Virgen del Rocío, Sevilla, Spain Background-aim Hyperphenylalaninemia (HPA) is the most frequent disorder of amino acid metabolism and is mostly due to recessive mutations in the gene that encodes phenylalanine hydroxylase (PAH). This enzyme works in the presence of the essential cofactor tetrahydrobiopterin (BH4). The HPA phenotypes are: mild hyper- phenylalaninemia (MHP or HPA III), mild phenylketonuria (MPKU or HPAII), and phenylketonuria (PKU or HPAI) depending of blood phenylalanine levels. The treatment is the dietary restriction, but an increasing number of PAH patients have been shown to benefit from BH4 supplementation. Aim: Characterize the genotype–phenotype and BH4 responsive- ness in 12 PKU patients form Western Andalusia (Spain). Methods Materials and methods: DNA was extracted from blood samples from dried blood spots (DBS) by the MagNA Pure system (Roche) Abstracts / Clinica Chimica Acta 493 (2019) S563–S583