Targeted Next Generation Sequencing in Patients with Inborn Errors of Metabolism

<div><p>Background</p><p>Next-generation sequencing (NGS) technology has allowed the promotion of genetic diagnosis and are becoming increasingly inexpensive and faster. To evaluate the utility of NGS in the clinical field, a targeted genetic panel approach was designed for the diagnosis of a set of inborn errors of metabolism (IEM). The final aim of the study was to compare the findings for the diagnostic yield of NGS in patients who presented with consistent clinical and biochemical suspicion of IEM with those obtained for patients who did not have specific biomarkers.</p><p>Methods</p><p>The subjects studied (n = 146) were classified into two categories: Group 1 (n = 81), which consisted of patients with clinical and biochemical suspicion of IEM, and Group 2 (n = 65), which consisted of IEM cases with clinical suspicion and unspecific biomarkers. A total of 171 genes were analyzed using a custom targeted panel of genes followed by Sanger validation.</p><p>Results</p><p>Genetic diagnosis was achieved in 50% of patients (73/146). In addition, the diagnostic yield obtained for Group 1 was 78% (63/81), and this rate decreased to 15.4% (10/65) in Group 2 (<i>X</i>² = 76.171; p < 0.0001).</p><p>Conclusions</p><p>A rapid and effective genetic diagnosis was achieved in our cohort, particularly the group that had both clinical and biochemical indications for the diagnosis.</p></div

Global genetic results.

<p>Genetic results (positive, under-study, and negative cases) shown as a percentages for each nosological group (aminoacidopathies (AA); organic acidurias (OA); free fatty acid oxidation defects (FFA); and neurometabolic (NM) and complex molecules (CM) defects) and for both diagnostic groups (Groups 1 and 2).</p

Triglyceride levels in skm, liver and adipose tissue of GFP- and FATP1-mice.

<p>Triglyceride levels were measured in extracts from the gastrocnemius muscle, liver and white adipose tissue of <i>ad libitum</i> fed GFP- or FATP1-mice. Data are means ± SEM of at least four samples. The significance of the Student's t test is: ^*p<0.05 and ^**p<0.001 female versus male GFP-mice fed chow; ^#p<0.01 female versus male FATP1-mice fed chow; ^†p<0.05 and ^††p<0.001 female GFP-mice fed high-fat versus chow; ^‡p<0.05 and ^‡‡p<0.001 female FATP1-mice fed high-fat versus chow; and ^¥p<0.05 FATP1- versus GFP-mice irrespective of diet and gender.</p

Glucose tolerance tests in GFP- and FATP1-mice.

<p>Blood glucose levels were measured before (0 min) and after glucose injection, several times up to 120 min, in GFP- or FATP1-mice fed as stated, chow or a high-fat diet (HFD). (A) Glucose data are shown and are means ±SD of five animals. (B) Glucose areas under the curve are shown and are means ± SD of five animals. The significance of the Student's t test is: *p<0.05 and ^#p<0.001 within FATP1- or GFP-mice fed a HFD versus chow.</p

Electron microscopic localization of FATP1-GFP in C2C12 myotubes.

<p>C2C12 myoblasts were transfected with (a,b) pFATP1-GFP or (c) pGFP (control), and induced to differentiate into myotubes. Four days post-transfection, myotubes were fixed, gelatin blocks mounted and sections were prepared in an ultracryomicrotome, incubated with anti-GFP antibody and analyzed by electronic microscopy. (a,b) Image of FATP1-GFP localized inside the mitochondria (see arrows); (c) image of GFP localized in the Golgi complex (see continuous arrows) and nuclei (dotted arrow). The observations were made in an electron microscope with a CCD camera and an electron accelerating voltage of 80 Kv was employed for the measurements. Bar represents 200 nm.</p

Body weight, blood metabolites and insulin levels in GFP- and FATP1-mice.

<p>Body weight, blood glucose, serum insulin, triglyceride, fatty acid and β-hydroxybutyrate levels were measured in <i>ad libitum</i> fed either diet GFP- or FATP1-mice at 15- to 16-weeks of age. Data are means ± SEM of at least five samples. The significance of the Student's t test is: ^†p<0.05 females fed a high-fat versus chow diet with the same viral treatment; ^*p<0.01 GFP-females fed a high-fat versus chow diet; ^#p<0.05 GFP-females versus GFP-males fed a chow diet; ^‡p<0.005 FATP1-females versus FATP1-males fed chow; and ^¥p<0.05 FATP1-mice versus GFP-mice irrespective of diet and gender. ND means not determined.</p

PDH levels and <i>Pdk4</i> expression in skm of GFP- and FATP1-mice.

<p>(A) Active PDH levels and (B) levels of phosphorylated PDH-E1α at sites 1 (P1) and 2 (P2) relative to α-actinin levels, were measured in extracts from the gastrocnemius muscle of GFP- and FATP1- female mice fed a chow or high-fat diet (HFD). Data are mean values ± SEM from at least four samples. The significance of the Student's t test is *p<0.05 and **p<0.01 in GFP-mice fed a high-fat versus chow diet; and ^#p<0.05 FATP1-mice fed high-fat versus FATP1-mice fed chow (C) <i>Pdk4</i> expression was analyzed in the gastrocnemius muscle of female GFP- and FATP1-mice fed as indicated.Data are mean values of 2^−ΔCp×10⁵ ± SEM from four to eight samples performed in duplicate. The significance of the Student's t test is: *p<0.01 in mice fed high-fat versus chow within the same GFP- or FATP1-condition; and ^#p<0.05 FATP1-mice versus GFP-mice fed high-fat.</p

Muscle <i>Hmgcs2</i> and <i>Oxct1</i> expression in GFP- and FATP1-mice.

<p>(A) The <i>Hmgcs2</i> mRNA levels relative to <i>Actb</i> and (C) <i>Oxct1</i> mRNA levels relative to <i>Rn18s</i> were analyzed in the gastrocnemius muscles of GFP- and FATP1-female mice fed a chow or high-fat diet (HFD). (A,C) The significance of the Student's t test is: *p<0.05 FATP1- versus GFP-female mice fed on the same diet; ^#p<0.05 control mice fed a HFD versus chow. <i>Hmgcs2</i> data are mean values of 2^−ΔCT ± SEM in arbitrary units from four to six samples performed in duplicate. <i>Oxct1</i> data are mean values of 2^−ΔCp×10⁵ ± SEM from four to six samples performed in quadruplicate. (B) The HMGCS2 and (D) OXCT1 protein levels relative to tubulin or α-actinin levels were analysed. A representative image is shown and bands were quantified. Data is the ratio of intensities of bands expressed as a percentage of control and is the mean ± SEM of at least four samples performed in duplicate.</p

β-hydroxybutyrate levels in skm and liver of high-fat diet fed GFP- and FATP1-mice.

<p>β-hydroxybutyrate levels were measured in extracts from the gastrocnemius and tibialis anterior muscle and the liver of GFP- and FATP1-female mice fed high-fat. Data are mean values ± SEM from four to five samples.</p

Effects of FATP1 overexpression on gastrocnemius muscle palmitate, β-hydroxybutyrate and glucose oxidation.

<p>Gastrocnemius muscle strips were prepared from pGFP- or pFATP1-electropored mice. (A) The FATP1 protein levels relative to α-tubulin levels were determined in total extracts and the 1500 <i>g</i> pellet fraction (20 µg of protein) by immunoblotting. Bands were quantified with a LAS-3000 (FujiFilm). Data is the ratio of intensities of bands in arbitrary units and is the mean ± SEM of at least five samples. (B) Muscle strips were incubated for 4 h with radioactively labelled palmitate (PA), β-hydroxybutyrate (BHB) or glucose (GLU). At the end of this period, substrate utilization rate, ¹⁴CO₂ production rate (and [¹⁴C]acid-soluble intermediate metabolites (ASI) production rate in palmitate-incubated strips) were determined. Data are expressed as a percentage of control and are the means ± SEM of seven muscle samples. (A,B) The significance of the Student's t test versus controls is *p<0.05.</p