Homozygous mutation in murine retrovirus integration site 1 gene associated with a non‐syndromic form of isolated familial achalasia

Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early‐onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease.


| INTRODUC TI ON
Achalasia is a rare disorder of esophageal motility characterized by esophageal aperistalsis and impaired relaxation of the lower esophageal sphincter (LES) during deglutition. The annual incidence of achalasia is approximately 1 in 100 000 people worldwide, with an overall prevalence of 9-10 per 100 000. 1 Up to now, several proteins involved in this process have been identified. However, there are still a number of unresolved cases with suggested genetic background.
The first described syndrome with achalasia was the triple A syndrome (MIM 231550) with the further main symptoms alacrima, adrenal insufficiency, and neurological impairment. 2 In this syndrome, the achalasia-addisonianism-alacrima syndrome gene (AAAS) is mutated. 3 This gene encodes a protein of the nuclear pore complex named ALADIN (alacrima achalasia adrenal insufficiency neurologic disorder). 4 Achalasia due to ALADIN dysfunction is predicted to originate by degradation of myenteric neurons of the LES due to increased oxidative stress. 5,6 In recent years, mutations in several new genes have been described in humans with a monogenic form of achalasia. These are genes encoding for proteins of the cG-MP-pathway such as nitric oxide synthase 1 (NOS1) and guanylate cyclase 1 soluble subunit alpha 1 (GUCY1A1; previous name: soluble guanylate cyclase 1 alpha 3; GUCY1A3) as well as genes with other cellular protein functions like cytokine receptor-like factor 1 (CRLF1), mitochondrial isoleucyl-tRNA synthetase (IARS2), stem cell factor receptor gene (KIT), trafficking protein particle complex subunit 11 (TRAPPC11), and guanosine diphosphate mannose pyrophosphorylase A (GMPPA). [7][8][9][10][11][12][13] Association studies led to the identification of an eight-amino-acid insertion in the cytoplasmic tail of HLA-DQβ1 as a major risk factor for disease onset of idiopathic achalasia suggesting autoimmune processes to be involved in the pathogenesis of achalasia. 14,15 Genes mediating esophageal motor function and gastrointestinal motility defects in mice such as the sprouty2 gene (SPRY2), myosin phosphatase target subunit 1 (MYPT1), and MRVI1 are suspected as candidate genes for achalasia in humans. 16 20 This process is regulated by nitric oxide (NO) that stimulates soluble guanylyl cyclases and increases cGMP levels. 21 The interaction of cGK1β, IRAG, and IP 3 R1 is essential for this regulation. 20,22 Decreasing the expression of IRAG in human smooth muscle cells has been shown to result in the abrogation of the inhibitory effect on calcium release from IP 3 -sensitive stores, which points to a physiologic role of IRAG in human gastrointestinal smooth muscle relaxation. 23 Here, we report a homozygous nonsense mutation in human MRVI1 associated with familial early-onset isolated achalasia in a Tunisian family with three affected siblings. The mutation causes loss of the interaction domain of IRAG with cGK1β and therefore contributes to the loss of its central role in calcium regulation supporting its pathogenic role.

| Whole genome sequencing
Blood samples from patients, parents, and healthy siblings were collected after informed consent for whole genome sequencing (WGS). DNA preparation was performed according to standard protocols using QIAamp DNA Blood Mini Kit (Qiagen). WGS was performed in two (II:1 and II:8) of the three patients of this family using the BGISEQ-500 pipeline, which generated 1252 Mio and 1403 Mio 100 bp paired-end FASTQ-encoded reads that were aligned to the human GRCh37.p11 (hg19) genome build using BWA-MEM v0.7.1 (http://arxiv.org/pdf/1303.3997.pdf). 24 The clean reads of the samples had high Q20 (96.26% and 97.09%) and Q30 (87.65% and 89.34%), which showed high sequencing quality.
A variant file for variants located in coding and flanking intronic regions was generated using the GATK v3.8 software package and sent to MutationTaster2 for assessment of potential pathogenicity (http://www.mutat ionta ster.org). 25,26 Variants were filtered using allele frequencies from gnomAD removing all variants with a minor allele frequency of >5E-5 for the dominant disease model and a homozygote frequency of 1E-4 for the recessive disease model. 27 All relevant variants were visually inspected using the IGV-software (http://www.broad insti tute.org/igv) and checked

Key Points
• Achalasia is a rare disorder of esophageal motility with an incidence of 1:100,000. Several proteins involved in achalasia have been identified, but there are still a number of unresolved cases with suggested genetic background. Using whole genome sequencing we investigate the genetic basis of early-onset isolated achalasia in a family of Tunisian origin with three affected siblings. for entry in the gnomAD database (http://gnomad.broad insti tute.org/), the Exome Aggregation Consortium (ExAC) database (http://exac.broad insti tute.org), the 1000 genome (1000G) database (http://www.1000g enomes.org) and the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP).

| Plasmids
Full-length MRVI1 cDNA of transcript variants 1 and 2 was cloned into pEGFP-C1 (BD Biosciences Clontech) in frame at the carboxy terminus of the enhanced green fluorescence protein (EGFP). For cloning of MRVI1 and mutagenesis of patient's mutation, we used the In-Fusion HD Cloning Plus kit (TaKaRa Bio Europe SAS) according to the manufacturer's instructions. Plasmid DNA was prepared using the QIAfilter™ Plasmid Maxi Kit (Qiagen GmbH) and verified by sequencing. cGK1β-dsRed plasmid used was from previous study. 22

| Cell culture and transfection
COS7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Biochrom AG) supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics, at 37°C and 5% CO 2 . The day before transfection, cells were plated in 24 well plates on cover slips at 5 × 10 4 cells per well or in 10 cm petri dish at a density of 2 × 10 6 cells in DMEM without antibiotics. After cultivation for 24 hours, cells were transiently transfected with 100 ng or 7,5 µg of each plasmid DNA using Fugene HD (Promega) or Polyethyleneimine (Sigma-Aldrich) respectively according to the manufacturer's instructions.

| Immunofluorescence analysis
Forty-eight hours after transfection, cells were fixed with 2% formaldehyde and permeabilized with 0.5% Triton X-100 for 5 minutes.
Blocking was performed with 3% BSA in PBS for one hour at roomtemperature (RT). Anti-Calnexin antibody (AF18, sc-23954; Santa Cruz Biotechnology Inc.) was diluted 1:50 in blocking solution and incubated at 4°C over-night in a humidified chamber. Secondary antibody Alexa Fluor 405 goat anti-mouse IgG (1:500) (Molecular Probes, Life Technologies) was incubated one hour at RT in the dark.
Excess antibodies after primary and secondary antibody staining were removed by fife washing steps using PBS. Cells on cover slips were attached on object slides with Vectashield ® mounting medium (Vector Laboratories). Confocal laser scanning was performed on an inverted Zeiss LSM 780 microscope (Zeiss) of the CMCB light microscopy facility. Images were acquired by setting the gain just below the threshold of signal saturation in blue, green, and red channels, and values for brightness, gamma, and contrast and brightness of individual color were adjusted equally by ZEN software.

| Affinity purification
Isolation of protein complexes consisting of cGK1β, IRAG, and IP 3 R1 from transfected COS7 cells by affinity chromatography was performed as described previously. 20,28 In brief, the cGK1β and IRAG co-transfected COS7 cells were solubilized in RIPA lysis buffer

| RE SULTS
To investigate genetic basis of a familial form of isolated achalasia, we performed WGS in two patients from a large Tunisian family. A full clinical information of all three patients is provided in Table 1 To explore whether the c.361C>T mutation in MRVI1 interrupts proper protein-protein interaction and subcellular localization of the protein, we used an in vitro test system because no patient material was available for functional analysis. It is known that the membrane protein IRAG which interacts with the soluble enzyme cGK1β is assembled in a ternary complex with IP 3 R1. 20 For visual analysis (immunofluorescence) and biochemical isolation of protein complexes (affinity chromatography experiments), we used COS7 cells, naturally expressing IP 3 R1, but not cGK1β and IRAG. 20      for the interaction of IP 3 R1 with IRAG and is essential for assembly of the IP 3 R1 within the cGK1-IRAG macro-complex. 18 The deletion of the IP 3 R1-IRAG interaction domain in IRAG D12/D12 mice is involved in the inability of cGMP to relax receptor-triggered phasic and tonic smooth muscle contractions. 18 In a similar manner, the MRVI1 mutation of the described family causes the loss of the interaction domain of IRAG with cGK1β, thereby disrupting the IP 3 R1-IRAG-cGK1 complex and presumably contributing to the loss of cGMP-regulated smooth muscle relaxation (Figure 4).

The interplay of smooth muscle cells (SMC) and interstitial cells
of Cajal (ICC) in nitrergic neurotransmission in the LES highlights disturbances in novel signaling pathways responsible for primary achalasia. It has been shown that basal LES tone is dependent on NO-sensitive guanylyl cyclase in both ICC and SMCs whereas nitrergic LES relaxation is predominantly mediated via ICC. 32 The extent of the described MRVI1 mutation in ICC function remains to be determined.

F I G U R E 2
Immunfluorescence analysis of co-transfected COS7 cells (GFP-IRAG green, cGK1β-dsRed red) reveal that wild-type IRAG (isoform a and d) is located at endoplasmic reticulum (ER-marker Calnexin; blue) and co-localizes with cGK1β (yellow arrows); co-localization on the right column (merged images). GFP-IRAGa Arg112* and GFP-IRAGd Arg121* are mislocalized in the cytoplasm (white arrow heads) and the nucleus (white arrows) and induce a partly mislocalization of co-transfected cGK1β (yellow arrow heads) IRAG is a 125-kDa membrane protein that resides in the ER We anticipate that the transcript variants 4 (NM_001100167) and 6 (NM_001206881) of IRAG encoding for a 597 amino acid protein (isoform c) of 66 kDa (NP_001093637) with a start codon in exon 9 of MRVI1 are not affected by the mutation in exon 4. It is conceivable that the identified mutation in exon 4 of MRVI1 is only relevant for specific tissues and therefore results in an isolated form of achalasia. Werder and colleagues described a tissue-specific alternative splicing with exon skipping and alternative splice donor and acceptor site usage. 34 They showed that COOH-terminally truncated IRAG variants lacking both the cGK1 phosphorylation and the IP 3 R1 interaction site counteract cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. 34 Recently, the polymorphism p.Pro186Ser (rs35857561) in MRVI1 was hypothesized to be a genetic susceptibility factor for moyamoya in European patients with neurofibromatosis type 1. 29  In conclusion, although MRVI1 mutations seem to be a rare cause of achalasia, testing a panel of genes associated with achalasia including MRVI1 should be considered in individuals with an early onset of the disorder.

D I SCLOS U R E S
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
KK, NR, and AH conceived and designed the experiments. KK analyzed and interpreted the data. DH recruited and clinically characterized patients. JS provided protocols and experimental expertise.
DL performed experiments. MS performed the bioinformatics analysis of sequencing data. KK and DH wrote the paper. All authors reviewed and approved the final version of manuscript.