A Novel Splice Site Indel Alteration in the EIF2AK3 Gene Is Responsible For

Sümegi et al. BMC Medical Genetics (2020) 21:61 https://doi.org/10.1186/s12881-020-0985-6 RESEARCH ARTICLE Open Access A novel splice site indel alteration in the EIF2AK3 gene is responsible for the first cases of Wolcott-Rallison syndrome in Hungary Andrea Sümegi1, Zoltán Hendrik2, Tamás Gáll1, Enikő Felszeghy3, Katalin Szakszon3, Péter Antal-Szalmás4, Lívia Beke2, Ágnes Papp3, Gábor Méhes2, József Balla1,5 and György Balla1,3* Abstract Background: Wolcott-Rallison Syndrome (WRS) is a rare autosomal recessive disease that is the most common cause of neonatal diabetes in consanguineous families. WRS is caused by various genetic alterations of the Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3 (EIF2AK3) gene. Methods: Genetic analysis of a consanguineous family where two children were diagnosed with WRS was performed by Sanger sequencing. The altered protein was investigated by in vitro cloning, expression and immunohistochemistry. Results: The first cases in Hungary, − two patients in one family, where the parents were fourth-degree cousins - showed the typical clinical features of WRS: early onset diabetes mellitus with hyperglycemia, growth retardation, infection-induced multiple organ failure. The genetic background of the disease was a novel alteration in the EIF2AK3 gene involving the splice site of exon 11– intron 11–12 boundary: g.53051_53062delinsTG. According to cDNA sequencing this created a new splice site and resulted in a frameshift and the development of an early termination codon at amino acid position 633 (p.Pro627AspfsTer7). Based on in vitro cloning and expression studies, the truncated protein was functionally inactive. Immunohistochemistry revealed that the intact protein was absent in the islets of pancreas, furthermore insulin expressing cells were also dramatically diminished. Elevated GRP78 and reduced CHOP protein expression were observed in the liver. Conclusions: The novel genetic alteration causing the absence of the EIF2AK3 protein resulted in insufficient handling of severe endoplasmic reticulum stress, leading to liver failure and demise of the patients. Keywords: Wolcott-Rallison syndrome, EIF2AK3 gene, Endoplasmic reticulum stress, PERK protein, Splice site variant, Indel alteration * Correspondence: [email protected] 1HAS-UD Vascular Biology and Myocardial Pathophysiology Research Group, Hungarian Academy of Sciences, 98, Nagyerdei krt, Debrecen H-4032, Hungary 3Department of Pediatrics, Faculty of Medicine, University of Debrecen, 98, Nagyerdei krt, Debrecen H-4032, Hungary Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sümegi et al. BMC Medical Genetics (2020) 21:61 Page 2 of 12 Background Methods Wolcott-Rallison syndrome (WRS, OMIM 226980) is a Patients and controls rare autosomal recessive disorder, first described in 1972 The index patient (Patient 1)andhersister(Patient 2) [1]. The leading clinical symptom of the disease is persist- showed the typical clinical signs and symptoms of Wolcott- ent neonatal diabetes mellitus that requires insulin treat- Rallison syndrome and expired due to multiple organ fail- ment. Diabetes develops typically not later than 6 months ure induced by viral infection at 4.2 years and 4 months of after birth, but delayed cases have been also described [1– age, respectively. Their parents were fourth-degree cousins. 5]. The poor prognosis of WRS is associated with liver The parents also have two younger twin sons without any failure triggered by endoplasmic reticulum stressors like signs of WRS. Additionally, the mother has two older viral-, bacterial infections, and hypoglycemia; later the daughters and one grand-daughter - from her former rela- outcome is determined by the possible complications of tionship – who are all healthy. Altogether 9 of the described insulin therapy and hepatic injury. The cause of death is 10 family members were subjected to genetic analyses multiorgan failure, liver and kidney insufficiency. The (Fig. 1a). The control samples used for immunohistochem- broad diversity of the clinical manifestation of WRS con- istry were the routine histological samples of a 43-year-old sists of spondylo-epiphyseal dysplasia resulting in growth female person, who died because of an acute fulminant in- retardation and short stature [1–6]. Developmental delay, carceration of truncus cerebri caused by an aneurysm rup- osteoporosis, anemia and neutropenia are rare manifesta- ture of the arteria communicans anterior. She had no other tions [1–6]. Only sporadic cases were described with diseases based on the autopsy results. hypothyreoidism [7], exocrine pancreas insufficiency [8, 9], skin [1] or central nervous system abnormalities DNA and RNA isolation (microcephaly, pachygyria, mental retardation) [10–13]. Genomic DNA and RNA were isolated from peripheral The genetic base of the disorder is well-characterized; blood of the still living Patient 1, living parents, children variants in the Eukaryotic Translation Initiation Factor 2- and a grandchild, using the QIAmp DNA and RNA Blood Alpha Kinase 3 (EIF2AK3) - or traditionally PKR-like Mini kits (Qiagen, Valencia, CA). Since the sister of the endoplasmic reticulum kinase (PERK) - are present in all index child died years before the diagnosis, formalin-fixed, of the cases [14]. The different forms of the altered gene paraffin-embedded liver tissues were used as source of may present different levels of gene expressions with vari- DNA in her case and the “Tissue protocol” of the QIAmp able extent of the protein function. Upon endoplasmic DNA mini kit (Qiagen) was applied to extract DNA. reticulum (ER) stress EIF2AK3 detects the accumulation of misfolded proteins in the ER, phosphorylates EIF2α and downregulates the rate of protein synthesis [15]. EIF2α Sequence analysis of the EIF2AK3 gene from DNA and phosphorylation results in the expression of CHOP, a RNA samples multifaceted transcription factor of ER stress. If the gene The coding regions and the exon-intron boundaries of is altered and the EIF2AK3 protein is absent or non- 17 exons of the EIF2AK3 gene were amplified in 18 functional, large amount of misfolded proteins are accu- PCRs. We designed the PCR primers by Primer 3 soft- mulating in affected cells and tissues. In cases of WRS the ware [18] with the exception of exon 3, where we used a alteration leads to proinsulin aggregation and β cells apop- primer pair reported in a previous publication [17]. tosis in pancreas. Other organs, mainly liver and kidney RNA isolated from white blood cells of Patient 1 and also demonstrate the signs of ER stress [16, 17]. her mother was reverse transcribed using the High- Our two presented cases - the first WRS patients in Capacity cDNA Reverse Transcription Kit (Thermo Fisher Hungary - had different longevity, although they were Scientific, Waltham, MA USA). We amplified the region siblings living in the same environment and treated by surrounding the observed genetic alteration from cDNA the same medical team. Their parents are fourth-degree using a special primer pair designed including sequences cousins of a family in Hungary with Romani (alias Roma) of exon 11 and exon 12. The PCR product was 201 bp. ethnicity. The disease was caused by a novel splice site Since DNA isolated from formalin fixed paraffin- variant resulting in a non-functional truncated protein, embedded tissue contains smaller, fragmented DNA, characterized at the DNA, RNA, and protein level. The special PCR reaction was designed surrounding the in- cause of their death was liver damage leading to multior- volved exon-intron boundary of exon 11. The PCR pro- gan failure at 4.2 year and 4 month of age. We were able vided a smaller product (129 bp). to document extreme endoplasmic reticulum stress even The PCR products were sequenced using a cyclic in the younger child. Our aim was to provide data on ER chain-termination sequencing reaction with the BigDye stress in WRS in order to address the hypothesis that ER Terminator kit (v.3.1 and v.1.1; Thermo Fisher Scientific) stress inhibitors could prove beneficial for patients until and the ABI PRISM 310 Genetic Analyzer (Thermo the en bloc liver-pancreas transplantation. Fisher Scientific), as described before [19]. Sümegi et al. BMC Medical Genetics (2020) 21:61 Page 3 of 12 Fig. 1 Genetic alteration in the members of the affected family determined by DNA analysis. Altogether 9 of the presented 10 family members were subject of the genetic analysis (a). Compared to the wild type sequence (b) Patient 1 was homozygous (c) while the mother (e), father, one of the brothers and one of the half-sisters were heterozygous carriers of the observed genetic alteration [g.53051_53062delinsTG (Ref Seq: NG_016424.1)] based on genomic DNA (“gDNA”) sequencing using DNA isolated from peripheral leukocytes. In the case of Patient 2, only formalin fixed paraffin-embedded tissues were available, therefore, DNA was isolated from these archived liver sections and the region of the variation was amplified by a special PCR. Sequencing of these amplified fragments proved that Patient 2 was also homozygous for the genetic alteration present in the family (d).

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