1 INTRODUCTION

Specific diagnosis of many genetic diseases is challenging. One area where greater difficulties are met is that of developmental disorders of the central nervous system, since many genes can be involved (van Loo & Martens, 2007) with many more to be discovered probably. In more broad terms, rare diseases (RD) are pathologies that affect less than 1 in 2000 people (Commission & – European Commission, 2020). Due to their low frequency, they present diverse difficulties, from the delay in the diagnosis to the lack of specific treatments. Most of them have a substantial impact on quality and life expectancy, affecting children and young people.

Since the diagnosis itself is the first obstacle that RD patients face, from which most medical decisions will rely on, it becomes of crucial importance to have a precise and accurate diagnosis. Molecular genomics approaches have helped in this sense. Next-generation sequencing (NGS) techniques have highly improved diagnosis of RD. Many studies have shown that the use of whole-exome sequencing (WES) in rare Mendelian diseases has a diagnostic yield between 30% and 50% depending on the disease (Clark et al., 2018; Yang et al., 2014). The use of whole-genome sequencing (WGS) in a cohort of pediatric patients has found causative genetic variants in 34% of the cases (Stavropoulos et al. 2016). These results encouraged our group to use these tools (both WES/WGS) to help with the diagnosis of pediatric patients with RD.

Here, we present the case of a 6-year-old boy presenting with severe encephalopathy with unclear cause, which started in the first months of life, whose etiological diagnosis was made by WGS.

2 METHODS

3 RESULTS

3.3 Frameshift variant in the PURA gene is a candidate pathogenic mutation

Results of our filtering rationale (see methods 4.2), are found in Table 1. An autosomal recessive inheritance for the patient's phenotype was plausible, since both parents were unaffected. For this reason, on the one hand, coding variants in homozygosity with low frequency were considered. Out of the 24 (21 non-synonymous variants) none of them were related to a gene with a concordant phenotype. Additionally, compound heterozygous variants were considered. Out of the 75 genes, neither of them was previously associated with disease, or the phenotype associated was not concordant to the patient's. Another possibility was a de novo mutation, appearing for the first time in a heterozygous state in the patient. For this reason, we investigated all heterozygous mutations in coding regions with very low frequency (<0.5%). Here, we found a frameshift deletion of 11 nucleotides (CCAGGGGGGCT, c.151_161del) (Figure 1a) in the only exon of the PURA gene (chr5:139493917–139493927, GRCh37, PURA:NM_005859:exon1:c.151_161del:p.P51fs) not previously reported in any of the population genomic projects (such as 1000 Genomes (The 1000 Genomes Project Consortium, 2015)/GnomAD (Karczewski et al., 2020) /ExAC (Lek et al. 2016)). The deletion occurs between the 51th and the 54th amino acid (the first 16% of the protein), it causes a stop codon at codon 147 after the deletion. Therefore, not only the reading frame is altered for 147 amino acids, but also it results in a 130 amino acid shorter protein. Additionally, the variant was evaluated with SIFT Indel 2 (Pauline, 2003) to predict its pathogenicity from an in silico point of view. It was predicted as “damaging” with a confident score of 0.858.

TABLE 1. Results of variant filtering

	1% hom	1% comp. Het	0.5% het	chrM
# variants	24	477	427	29
# genes	14	75	196	12
Missense	19	273	373	9
Nonsense	1	10	14	0
Stoploss	0	1	1	0
fs indel	1	15	17	0
Non-fs indel	0	2	0	0
Silent	3	162	0	18
Splicing	0	3	5	0
Others	.	Unknown 10	Unknown 17	2 tRNA

• Columns show each of the four filtering categories: homozygous variants with a population frequency of less than 1%; heterozygous variants with a population frequency of less than 1%, with at least two variants in the same gene; heterozygous variants with a population frequency of less than 0.5%. Rows have the total number of variants found and the number of genes involved, and below the different types of variants found in each category. Unknown category corresponds to various ambiguities in the gene structure definition in the database file. fs, frameshift; non-fs, non-frameshift. # symbol represents number.

4 DISCUSSION

The PURA gene is a DNA/RNA-binding protein which functions as a transcription regulator also involved in mRNA localization (Weber, 2016). A number of pathologies, ranging from encephalopathies and mental retardation to leukemia and ALS, have been associated with heterozygous mutations in the PURA gene (Daniel, 2018; Johnson & Gordon, 2013). Its expression is ubiquitous with no clear tissue preference (human protein atlas, www.proteinatlas.org). The gene product is a protein with three domain repeats: repeats I and II are binding domains and repeat III is a dimerization domain (Johnson & Gordon., 2013; Weber, 2016). The crystal structure of the first two domains involved in nucleic acid binding from D. melanogaster complexed with ssDNA, has provided definitive evidence into the molecular interactions of the PURA protein (Weber, 2016), proving its purine base preference allows it for binding both DNA and RNA.

Mutations in the PURA gene were reported as a cause of dominant form of severe delayed psychomotor development and seizures a few years ago (Hunt et al., 2014; Lalani et al.,2014; Tanaka et al., 2015) and the PURA protein was shown to be a key player in brain development. PURA gene knockdown (−/−) mice show severe neurological and brain developmental anomalies and die within 4 weeks after birth (Hokkanen, 2012; Khalili et al., 2003).

Several lines of evidence point to the variant we found as a causative mutation. The change is predicted to be harmful to protein function, since it falls in the first quarter of the protein (AA 51 to 54 of 323 AA) producing an altered reading frame (for 147 AA following the deletion) and generating a premature stop codon (product is 130AA shorter), which leads to two possibly overlapping scenarios. On the one hand, the production of prematurely truncated mRNAs to be processed by the nonsense-mediated mRNA decay pathway (Brogna, & Wen, 2009). On the other hand, the production of a different protein, which we posit would be 195 amino acids: PURA gene's first 50 residues followed by a different sequence starting position 51, and therefore, affecting all of the three functional domains of the protein. This predicted protein has no detectable eukaryotic homologs, as checked by an HHpred (Söding, 2005) search over the Protein Data Bank or two iterations of jackhammer (Potter et al., 2018) over the Reference Proteome database. The production of an abnormal protein may result in multiple cellular problems, including accumulation of misfolded proteins. In this sense, the most probable transcript, if translated, is predicted to have high disorder propensity. Any of those mechanisms probably causes a total loss of function (LoF) of the product encoded by this allele. LoF has been demonstrated to be the main pathogenic mechanism in diseases associated with this gene (Bonaglia et al., 2015 and see below). Furthermore, microdeletions involving the PURA gene are also associated with a form of dominant epileptic encephalopathy (Hosoki et al., 2012; Shimojima et al., 2011). In most of the cases, these are de novo mutations with apparently complete penetrance.

Additionally, the variant fulfills following ACMG criteria for pathogenesis: (a) PVS1 (pathogenic very strong), since the deletion is a null variant in a gene for which LoF is a known mechanism of disease. According to the Varsome database (Kopanos et al., 2018) PURA gene has 70 pathogenic LoF variants and a significant LOF Z-Score of 2.77, associated with Mental retardation, autosomal dominant. (b) PS2 (pathogenic strong), since it is a de novo mutation (both maternally and paternally confirmed) in a patient with the disease and no family history. (c) PM2 (pathogenic moderate), since the variant has not been previously reported in patients nor it has been described in GnomAD and/or other large-scale population studies involving healthy individuals (the region has a good coverage in GnomAD (>25x) according to Varsome analysis). iv) PP3 (supporting evidence of pathogenicity), since an in silico score (GERP) predicted the variant as pathogenic (and no score predicted it as benign). Hence, all together the variant classifies as pathogenic according to ACMG.

There are several reported frameshift mutations in nearby codons (40, 46, 54, 59, and 83) as well as nonsense mutations (e.g., p. Gln55Ter, see https://www.ncbi.nlm.nih.gov/clinvar/variation/426145/) in that region of the protein, all of them reported as pathogenic. Other nonsense variants downstream in the protein (p. Gln163*, p. Gln186*, and p. Tyr261*) have also been reported as pathogenic (see: https://www.ncbi.nlm.nih.gov/clinvar/variation/582890/).

Finally, segregation analysis in the family (parents) supports a de novo mutation hypothesis which is consistent with both parents and half-siblings being healthy. In addition, no other variants in the PURA gene of less than 1% population frequency have been detected in this patient.

There are several patients reported with gene disrupting mutations which likely cause haploinsufficiency (nonsense and frameshift indels). In a larger series of patients reported so far (Reijnders et al., 2018 and Lee et al., 2018), no significant differences in clinical severity between mutation classes was found. As expected, in our patient, whose mutation causes a complete loss of function of the affected allele, whether caused by protein primary structure disruption or/and truncation or nonsense-mediated mRNA decay, the clinical presentation is severe with serious defect in motor skills, absence of language, respiratory problems, and intense epileptic activity.

Therefore, we think that the variant we found is a strong candidate pathogenic mutation probably explaining the patients’ phenotype marked by severe central neurological dysfunction.

CONFLICT OF INTERESTS

Authors declare no conflict of interests.