Phenotype and genotype characteristics of neonatal onset inflammatory bowel disease with combined immunodeficiency of TTC7A deficiency in mainland China


 Objective: To explore the characteristics of genotype and phenotype of neonatal onset inflammatory bowel disease with combined immunodeficiency caused by a novel TTC7A mutant. Methods: We summarized the clinical manifestations, imagings, endoscopic and histological findings, biochemical analyses,whole exon sequencing(WES), in silico and intervention of the patient. Results: The boy showed severe diarrhea, malnutrition, electrolyte disturbance, dehydration and recurrent infections after birth. X-ray and ultrasonic images displayed no specific changes. Endoscopic and histological findings showed chronic inflammation. Immune functions indicated combined immunodeficiency. WES identified compound heterozygote TTC7A mutations c.2355+4A>G/c.643G>T in the infant. No abnormal splicing sequence by c.2355+4A>G mutation was found in TTC7A expression analysis, but the mRNA expression decreased. There was no improvement after treatment with methylprednisolone and leflunomide. The infant died when he was given up at 5 months 19 days old. Conclusion: The compound heterozygote mutations (c.2355+4A>G, c.643G>T) in TTC7A gene were firstly described and confirmed. Our report expands the phenotypic spectrum of TTC7A mutaions and genotypic spectrum of very early onset inflammatory bowel disease with combined immunodeficiency.

The proband, an infant male, was born to non-consanguineous Chinese parents at 35 weeks and 6 days of gestation by in vitro fertilized. His mother was a 27-year-old healthy female and his father was a 29-year-old healthy male. No complication was detected during prenatal period. His birth weight was 2800g, and body length was 50cm. He was breastfed and presented diarrhea with yellow, watery and bloody stool for 5-10 times per day after birth. He was admitted to a tertiary Children's hospital, but received no improvement after 1 week of anti-infective treatment. For better treatment, he was admitted to our hospital at the age of 1 month and 8 days with weight 2600g, body length 50cm, and head circumference 33cm. No special facies and deformity were found. He showed malnutrition, moderate dehydration, and severe perianal dermatitis.
Because of the large quantity of watery stool (about 300-940mL per day), he was given total parenteral nutrition, amino acid milk powder, deep hydrolyzed protein milk powder, or breast milk feeding after admission, but without any improvement. The watery stool test showed sodium was 99.4 mmol/L(serum reference 135-145 mmol/L), potassium was 31.3mmol/L(serum reference 3.5-5.5mmol/L), and chloride was 109.8mmol/L(serum reference 99.0-110.0mmol/L), which was considered as high output secretory diarrhea.
During hospitalization, recurrent fever appeared and the detection of immune function indicated CID (Table 1). Cytomegalovirus antibody test was negative, but cytomegalovirus DNA in blood was 4.12*10 6 copies/mL and 1.47*10 6 copies/mL in urine.
Cytomegalovirus and pneumocystis carinii were found in bronchoalveolar lavage fluid by metagenomic detection, and candida albicans in urine and sputum cultures.

Image features, endoscopic and histological characteristics
The patient underwent abdominal X-ray, ultrasound, gastroscopy, colonoscopy and      There were no abnormal splicing bands in agarose gel electrophoresis, and no abnormal splicing sequence was found in Sanger sequencing. However, the expression of TTC7A decreased in the proband, which may lead to the decrease of protein expression.
While no abnormal expression of TTC7A gene was detected in the parents (Figure 6-8).

Discussion
IBD is a global disease which has an overall incidence rate of 37.5/100,000 person yearly and was highest in adults and the elderly, and lowest in pediatric (0-17 years) in the United States from 2005 to 2015 [18]. In pediatric IBD group, VEOIBD accounted for about 3%, and infantile onset IBD accounted for only 1%, the incidence of VEOIBD remained stable, however the incidence of older children increased [19]. These results may suggest a probable genetic origin for VEOIBD, whereas the increase in older children might be linked to environment factors [19]. A most important clinical sign is young age of onset in monogenic IBD. In our study, the patient was first thought to be related with genetic factors, because of his very early onset and severe symptoms.
Neonatal or infantile onset IBD is prone to suffer from primary immune deficiency disorders especially caused by mutations of IL-10, XIAP, NCF2, IPEX, and TTC7A gene etc [10]. TTC7A gene has 20 exons and includes 9 TPR domains, and mutations in exon 2, exon 7 and exon 20 are relatively common [20]. TPR domains are helix-turn-helix structurally conserved motifs as an important role in protein scaffolding and have large surface areas which accommodate multiple-protein interactions [21,22]. No case of TTC7A gene mutation has been reported in mainland China till now. Our case confirmed two novel TTC7A gene mutations, c.643G>T in exon 4 from his father and c.2355+4A>G in exon 19 from his mother, which have not been previously described in HGMD. The c.643G>T, a nonsense mutation in exon 9, results in a premature termination of the protein and is predicted to cause a loss of function. The c.2355+4A>G mutation was demonstrated to decrease the mRNA expression by qPCR. We once thought that this intron mutation would affect splicing, but interestingly, the experimental results were not the case. Few researches have ever performed functional analysis of specific variants up to now. Protein analysis via immunostaining or immunoblotting was not detected. We speculate the c.2355+4A>G mutation may affect TPR domain which binds and recruits phosphatidyl inositol 4 phosphatidylinositol 4-kinase III alpha to the plasma membrane, facilitating the synthesis of PI4-phosphate Mutations affecting TPR domains tend to cause worse phenotypes and poor outcomes [23]. Some studies of truncating TTC7A mutations suggested nonsense-mediated decay of TTC7A messenger RNA transcripts and showed obvious loss of protein consistent with the increased phenotypic severity in patients with MIA-CID whereas nontruncating mutations tend to present as VEOIBD [12,13]. Although nonsense mutations were thought to cause severe intestinal diseases (e.g MIA) and CID, missense mutations mainly present with IBD and lighter intestinal disease [24,25]. Our patient not only has truncated mutation, but also affect the TPR domain, which manifests as neonatal onset IBD and CID ， not MIA. Complex phenotypes seem difficult to attribute to physical alternations on the TTC7A gene.
Less than 10% patients of TTC7A mutation manifest as VEOIBD with secretory diarrhea, chronic intestinal inflammation, lymphocytopenia, and/or hypoglobulinemia, etc [15,20]. VEOIBD is more likely to presents with isolated colonic involvement and paucity of ileal involvement. Rectal bleeding and mucous stools with weight loss and underlying primary immunodeficiency were more common in VEOIBD group of monogenic causes [1,19,26]. X-ray, and ultrasound are the most commonly useful methods of examination in pediatrics, especially before the diagnosis of IBD. No specificity and significant difference was found between pediatric and adult IBD patients [27]. Imaging studies of VEOIBD were relatively rare. Hepatosplenomegaly, mildly dilated large and small bowel, significant bowel wall thickening under ultrasonography were reported in neonatal IBD [28]. In our patient, abdominal X-ray revealed stiffness in the intestines and decreased intestinal gas. Gastrointestinal ultrasonography showed thickening of the bowel wall, reduction of intestinal gas, and fluid accumulation in the colon. All these imaging examinations were consistent with the pathologic changes of apoptosis, twisted crypts and hypersecretion of mucus in colon. It is very difficult to perform gastroenteroscopy in early neonatal or infant period because of severe conditions with a high risk, poor bowel preparation, and technical failure. The patient underwent gastroenteroscopy at about 3 months old, and chronic mild superficial inflammation changes in esophagus, stomach and duodenum, twisted crypts and hypersecretion of mucus in colon were found, unlike atypical changes of older children or adult ulcerative colitis or Crohn disease, which mainly involve the colon. More pediatric IBD patients have extensive ileocolonic inflammation compared with the adult group [29].
The most common endoscopic findings were mucosal bleeding among VEOIBD patients, and visual ileitis or ileocolonic in older onset IBD patients [30]. Increased frequency of apoptosis, severe chronic structural changes, blunt villi in the small intestine, and eosinophils in the crypts, lamina propria, and surface epithelium reported in VEOIBD, were also discovered in some primary immunodeficiencies [30]. T-cell maturation disruption and lymphocytopenia because of hypoplastic thymus, and low blood immunoglobulin levels owing to increases in the relative levels of transitional B cells and B-cell receptor assembly and organelle synthesis in activated B cells were speculated [13,31]. Therefore, we speculate that immunodeficiency is closely related to VEOIBD, and immune function should be tested as soon as possible. According to the data of our case, the phenotype involves extensive lesions in stomach, duodenum, small intestine, and colon, leading to very early onset and severe symptoms, and immunodeficiency.
Most monogenic IBD patients are resistant to conventional medical treatment [1,16,26], so is neonatal onset IBD of TTC7A deficiency. At present, treatments of manifestations, including early intestinal resection (in MIA), parenteral nutrition, regular immunoglobulin infusion, were recommended [32]. Total or partial parenteral nutrition was reported in many TTC7A patients [13,14,33]. Because of intestinal malabsorption and excessive loss, early application of parenteral nutrition support is necessary. In our study, parenteral nutrition support started very early.
Hematopoietic stem cell transplantation was reported in monogenic IBD, which may restore immunity and increase survival in immunodeficiency patients, but it does not seem to improve phenotypes associated with intestinal epithelial defects [15]. The survival rate of TTC7A deficiency treated with hematopoietic stem cell transplantation was not improved [32,34]. Gilroy et al found intestinal and liver transplantation restored intestinal function and immune function in a child with MIA-CID of unknown genetic etiology [35]. Unfortunately, intestine transplantation is not feasible due to the limitation of donor factors. In vitro studies have proved that Rho A inhibitors (Y-27632) and leflunomide were effective [36,37], but according to reported cases, no TTC7A-deficiency patients have received leflunomide. Our patient accepted leflunomide for 4 weeks after obtaining the consent of the parents, but it did not work. The efficacy of leflunomide in vivo remains uncertain. This infant died when he was given up at 5 months 19 days old.
. Genotype of TTC7A deficiency may be closely related to prognosis. An average survival age in patients of TTC7A gene nonsense mutations was less than 12 months. Some patients with biallelic missense mutations not involving TPR domains and autoimmune disorders had a relatively better prognosis and survived to adulthood [24,25].

Conclusion
In summary, two novel compound heterozygous mutations in TTC7A gene are firstly identified in mainland China. It manifested as neonatal onset IBD and combined immunodeficiency with poor prognosis. No specific manifestations were found in imaging examinations and endoscopic findings. The study of the genotype and clinical phenotype of TTC7A deficiency needs to accumulate more clinical data and conduct in-depth functional research.