Introduction

Adenylosuccinate lyase deficiency is a rare inherited neurometabolic disorder of purine metabolism with the combination of neurological and physiological symptoms such as psychomotor retardation, epilepsy, autistic behaviors, respiratory failure and Muscular hypotonia (1,2). For the first time in 1984, Jaeken and van den Berghe described it by finding succinylpurines in the cerebrospinal fluid (3).

ADSL deficiency is classified into three fatal to mild forms with wide spectrum of signs and symptoms. Patient's phenotypes have classified as severe form type I, milder type II and fatal neonatal form (4). The type I form is characterized by microcephaly, severe neurodevelopmental delay, seizures, and autistic features and most of the patients reported so far have ADSL type I (5). Type II form include moderate psychomotor delay and seizure and Fatal neonatal form instead is encephalopathy, Refractory seizure and respiratory failure and leads to early death (6).

The prevalence has been estimated 1 in 1.25 million and up to now less than 100 cases have been described in the world (7).

Here, we represent a five-and-a-half-year-old boy who referred to our neurometabolic clinic. He had been diagnosed as having ADSL deficiency with no obvious signs of disease progression and degradation.

Case Study

A five-and-a-half-year-old boy from Qazvin was transferred to Mofid Hospital, Shahid Beheshti University of Medical Sciences with Refractory seizure and Neurodevelopmental delay (NDD).

His parents were cousin and he was 4th child of a consanguineous marriage.

One of his older brother who was the second child in family suffered from NDD with refractory seizure and passed away at the age of 3.5 years old.

The patient was bedridden and has suffered from tonic seizure 3 to 10 times a day since 3 months of life.

He could have head control until being 3 months old and there after his general developmental condition seemed to progressively delayed; and now global developmental delay is obvious in physical and mental examination.

Different type of antiepileptic drugs had been prescribed due to seizure reduction, but without obvious control.

His neurological exam showed hypotonia, negative fix and follow with normal DTR and Magnetic resonance imaging (MRI) revealed evidence of Ventriculomegaly and Front temporal atrophy.

Hypomyelination, enlarged lateral ventricles and cortical atrophy

MRI shows delayed myelination, enlarged lateral ventricles, abnormal white matter signal, and volume loss throughout the cerebrum or cerebellum which is apparent in the first few months of life and radiologist may be the first individual to prompt this disease.

After neurological evaluation and laboratory investigations assessment, patient referred for      genetic testing.

Genetic investigation

To investigate the genetic cause, whole blood samples were collected from patient and his parents in EDTA containing tubes. Genomic DNA was extracted from whole blood using Blood SV-mini kit (GeneAll Biotechnology Co., LTD, South Korea) according to the manufacturer instruction.

DNA sample quality control was performed before using DNA for whole exome sequencing. Quantity of DNA was measured by PicoGreen (Invitrogen, Thermo Fisher Scientific, USA) method using Victor3 fluorometry as well as a micro-spectrophotometer. Assessment of DNA condition was performed by gel electrophoresis.

Library preparation was performed using Agilent SureSelect Human All Exon V7 system (Agilent Technologies Inc.,Santa Clara, CA, USA) using manufacturer instruction. Sequencing of libraries was done by high-throughput paired-end sequencing using HiSeq2000 sequencing platform (Illumina Inc., CA, USA).

Sequencing short reads were aligned to the reference human genome hg19 from UCSC genome browser (University of California, Santa Cruz, USA) via the Burrows-Wheeler Aligner (BWA) program. Variant calling and filtering was done using Genome Analysis toolkit (GATK-v3.4.0). Detected variants were annotated using WANNOVAR software.

Proper filtering and then interpretation of a short list of variants in terms of pathogenicity was performed based on ACMG (American College of Medical Genetics and Genomics) guideline for variant interpretation.

To evaluate the pathogenicity of candidate variants, the potential impact of a given variant on the function or structure of the encoded protein was analyzed. The analysis was carried out based on conservation, physical properties of the amino acids or possible occurrence in regulatory or splicing motifs using bioinformatics tools. OMIM and PubMed was reviewed for previous publications related to the candidate causative gene.

After defining a pathogenic variant as the causative variant in the patient on WES data, patient’s homozygous genotype as well as carrier status (heterozygosity) of his parents was validated by PCR (Polymerase Chain Reaction) and Sanger sequencing using ABI 3500 Genetic Analyzer (Applied Biosystems Inc., CA, USA).

Results

Analysis of WES data ends up with a homozygous variant in exon 12 of ADSL gene on chromosome 22 (c.1277G>A, p.R426H, hg19: 22-40760969-G-A).  Homozygosity of variant in patient was also confirmed by PCR-sanger sequencing (Figure 1-a).  Parental analysis confirmed heterozygous carrier states in his parents (Figure 1-b-c) that is consistent with the autosomal recessive pattern of inheritance. Having these finding in addition to bioinformatic analysis, detected variant was classified as a pathogenic variant based on ACMG guideline for variant interpretation. Having this classification, diagnosis of disease is confirmed in our patient.

Figure 1: Homozygosity for variant c.1277G>A in ADSL gene in patient (a) and heterozygosity in his mother (b) and his father(c) was confirmed by PCR-Sanger sequencing.