Metabolic profiling of organic acids in urine samples of Cri Du Chat syndrome individuals by gas chromatography-mass spectrometry

Highlights

• Organic acids highlight biochemical alterations caused by Cri Du Chat syndrome.

• Metabolites analyzed by GC–MS shows high intensities and good reproducibility.

• Metabolomics approach points out changes in the urinary organic acid levels.

• Altered metabolic pathways indicate neuropsychiatric condition due to the syndrome.

Abstract

Cri Du Chat (CDC) syndrome is a rare genetic condition caused by the deletion of genetic material on the small arm (the p arm) of chromosome 5. A high-pitched cry that sounds like that of a cat, dysmorphic characteristics, and cytogenetic methods are often used for diagnosing the syndrome. In this study, we applied GC–MS analysis for determining organic acids in urine from 17 control volunteers without CDC syndrome, and from 16 individuals with the CDC syndrome in order to determine the profile of organic acids and biochemical pathways alterations resulting from this genetic condition. First, performing multivariate data analysis selected the best method for extracting organic acids with greater signal intensities and good reproducibility. After selection, multivariate (PLS-DA) and univariate (Mann-Whitney test) data analysis discriminated the metabolites responsible for separation between groups. Nine organic acid metabolites had values of VIP ≥ 1.0 and p-values ≤ 0.05, with highest intensities in the samples from CDC individuals, indicating the strongest discriminative power (tricarballylic acid, indoleacetic acid, anthranilic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 4-hydroxyhippuric acid, pantothenic acid, homovanillic acid, and vanillylmandelic acid). These metabolites are involved in several biochemical pathways like in the tyrosine and phenylalanine metabolism, as well as the tryptophan metabolism, which could be associated (i) to some neuropsychiatric alterations commonly observed in CDC individuals, (ii) to exogenous compounds related to transformation products by intestinal microbial, and (iii) to a possible deficiency in enzyme activity due to the syndrome.

Introduction

Cri Du Chat (CDC) syndrome or 5p-syndrome is a genetic condition caused by the deletion in the small arm (the p-arm) of the chromosome 5. CDC is classified as a rare condition occurring in one out of every 20,000–50,000 live births [1], which was first described by Lejeune et al. [2] who described a craniofacial dysmorphism (microcephaly) with a phonetic peculiarity reminiscent of a cat crying [2]. Intellectual disability, late development, abnormalities in the head (such as microcephaly), round face, hypertelorism, low birth weight, and weak muscle tone (hypotonia) are often observed in the CDC syndrome clinical characteristics [3], [4].

The accurate diagnosis of this genetic condition is basically carried out by karyotyping, classical and molecular cytogenetics methods,– and clinical characteristics of the CDC individuals [3], [5].

Some studies have been shown the relation of genotype-phenotype in CDC syndrome, as the sound of a cat crying correlated to a specific deletion in the 5p15.3 region [6], speech delay, and facial features [7] with 5p15.32 and 5p15.2, respectively. In addition, common clinical symptoms related to CDC carriers might also involve codified proteins by the chromosomes 1 and 2; meanwhile, genes located in the 5p- chromosome were correlated to the functions of metabolism, to the cellular cycle, tissue development, and to the immune system [8].

Inherently, to understand the biochemical changes intrinsic to such CDC genetic condition, it is believed that metabolic information can help to better understand the dynamic molecular changes that occur due to the different genetic profiles of individuals (such as those with CDC syndrome versus controls without CDC syndrome). This approach opens an opportunity to increase the knowledge of how diverse metabolic pathways are altered due to deletions in the genetic material. As an example, changes in purine synthesis and disorders in the amino acid catabolism have already been observed due to the partial deletion on chromosome 5p [9], [10], disturbers of redox balance, energy recuperation, branched chain amino acids [11] and glycolysis [12] have already been described in CDC individuals.

Metabolomics is a recent field of the omics sciences that aims the comprehensive characterization of small molecules (<1500 Da) called metabolites in biological systems [13]. It has been defined as an appropriate approach to provide information about the interactions between the genome, the transcriptome, and the proteome with the external environment, which enables a comprehensive understanding of cellular functioning as nutrient absorption and distribution, detoxification, transduction, and signal regulation, as well as information on metabolic processes, highlighting anabolism and catabolism [14], [15].

Metabolites have different chemical classes such as amino acids, carbohydrates, nucleotides, fatty acids, and organic acids; they dynamically participate as intermediates and products of several biochemical reactions mediated by enzymes in a living organism. The qualitative collection of metabolites present in biological fluids, cells, organs, and tissues is called metabolome [16]. Among the chemical classes mentioned, organic acids have gained scientific interest because they are intermediate metabolites and products in several critical metabolic pathways, such as the Krebs cycle, carbohydrate metabolism, ketone body metabolism, fatty acid oxidation, turnover of neurotransmitters, and protein metabolism [17].

For the analysis of organic acids, gas chromatography and mass spectrometry (GC–MS) have become powerful tools on metabolomics studies because they provide high resolution in separation, present reliable and reproducible mass fragmentation patterns for an wide range of metabolites, as well as high capacity for the identification of unknown compounds belonging to a metabolome [18], although previous chemical modification of the compounds (derivatization) is needed to extend the application range of GC–MS analysis. One of the most common applications of GC–MS is the monitoring and diagnosing of inborn errors of metabolism resulting from enzyme deficiencies, in which organic acids are usually the targets [19], [20], [21].

In the present work, we chose to carry out a metabolic profiling metabolomics approach focusing on the organic acids in urine samples from CDC individuals and other volunteers without this genetic condition. Profiling the organic acids and others altered metabolic pathways in CDC syndrome using different organic acids extraction methods helped to sort out both groups and identify the relevant metabolites.