Acquired Pure Red Cell Aplastic in one of the Monozygotic Twins with Common Variable Immunodeciency

Background Common variable immunodeciency (CVID) is the most prevalent primary immune defects in has become clear that most cases of CVID have a polygenic rather than a monogenic inheritance. CVID patients are prone to recurrent infection, and an increased incidence of certain autoimmune and neoplastic disorders. Monozygotic twins share identical genetic basis and may serve as a powerful model for study on genetic defects. Mutation-Taster (D, disease-causing; N, polymorphism); CADD (score > 10 implied deleterious variations); ACMG (U, uncertain signicance; B, benign; PM1-6, pathogenic moderate; PP1-5, pathogenic supporting; BS1-4, benign strong; BP1-6, benign supporting.)


Background
Common variable immunode ciency disorder (CVID) is one of the most prevalent primary immunode ciencies with an important morbidity and high number of medical encounters. Majority of patients are predisposed to recurrent and severe infections as a result of immune system failure. A signi cant minority suffer from autoimmune disease, sarcoid-like granuloma or malignancy [1]. The reported prevalence is about 1:50,000-1:20,000. Onset can vary from 4 years old to late adulthood [2].
Mortality rate in the rst 10 years after diagnosis ranges from 10%-35% in CVID patients [3]. The current CVID diagnostic criteria were developed by International Consensus Document (ICON) in 2016, as follows: the IgG level is repeatedly lower than local reference range; IgA or IgM level also is low; poor response to at least 1 type of antigen; other causes of hypogammaglobulinemia have been excluded [4].
Most of CVID cases occur sporadically, about 5%-25% of patients have a familial tendency, of which most demonstrate an autosomal dominant inheritance [5]. So far, monogenic causes have been identi ed in 2-10% of affected individuals with both autosomal recessive and dominant inherited, PIK3CD and LRBA gene mutations comprise approximately half of published cases, while more than 30 additional genes have also been reported. It has been recently suggested that, apart from rare monogenic forms, CVID is a complex rather than a Mendelian disease [6]. Whole exome sequencing (WES) is a potential effective tool to help discover genetic defects, which targets protein-coding sequences accounting for 1% of the whole genome, but reported to harbor 85% of disease-causing variants [7]. WES is commonly used to explore the pathogenic genes of CVID [8,9].
Here we describe the phenotypes of a twin with CVID. Extensive phenotypic pro le of circulating B and Tcell subsets were obtained by ow-cytometry at diagnosis and during several years follow-up. WES allowed us to identify a combination of variants with putative impact in the immune system and support a polygenic basis for the CVID phenotype and their concordant immune evolution.

Patient samples
Case 1 (proband): a 30-year-old male with a history of 7-year recurrent chronic watery diarrhea, 1-year lower limb weakness and back pain. The result of bone mineral density (BMD) test suggested severe osteoporosis with increased serum alkaline phosphatases (ALP) and parathyroid hormone (PTH).
Multiple times of colonoscopy examination indicated that chronic mucosal in ammation and interstitial edema, lymphoid proliferation in the submucosa and multiple lymphoid follicles, no more obvious abnormality was found. Low levels of all immunoglobulin types were determined by nephelometry.
Peripheral blood ow cytometry analysis revealed a low proportion of CD19 + cells and CD4+/CD8 + ratio. In January 2017, he suffered with acute severe anemia with normal level of mean corpuscular volume.
Reticulocyte count is 5000/µl (< 10000/µl). Bone marrow evaluation of the patient showed the nucleated erythrocyte was only 1.5% (< 5%) with normal granulocytic and megakaryocytic line age, and early erythroblast was observable as shown in Fig. 1b. Cytogenetic evaluation showed normal male karyotype.
Flow cytometry revealed no evidence of a lymphoproliferative disorder and other hematology diseases. Chest computed tomography scan ruled out the presence of thymoma, and there were no evidence of active HIV or viral hepatitis or other viral infection. Autoimmune antibodies were negative. Therefore, the diagnosis of acquired PRCA was made. Hemoglobin recovered after 5-month's treatment with low dose (100 mg/d) of cyclosporine. And then, the maintenance treatment lasted one year. Case 2: the proband's monozygotic twin brother, who also suffers recurrent respiratory infection and occasional diarrhea for several years. He developed into intermittent claudication due to bone pain in lower limbs since 2016. Symptoms gradually worsen and eventually lead to inability to walk. BMD test suggested severe osteoporosis with increased serum ALP and PTH, low levels of serum immunoglobulin and CD19 + cells proportion.
The twins have no protective titers of antibody to Hepatitis B virus, cowpox vaccines. Intravenous immunoglobulin replacement and oral vitamin D signi cantly relieved the symptoms of infection and bone pain in the twins. Their clinical characteristics are shown in Table 1 and both met the criteria for CVID [4]. Except an uncle (II:2) of the twins had leukemia, all of the members of their family, including their parents, were CVID-symptoms free and had normal immunoglobulin level. Father's BMD test is normal. But mother's BMD is lower than peers. The pedigree of the family is shown in Fig. 1a. The twins and their unaffected parents were recruited for the experiment. All participants provided written informed consent and the study was approved by the Shanghai Jiaotong University School of Medicine Renji Hospital ethic review board.

Whole exome sequencing
Genomic DNA of the twins and their parents was isolated from peripheral blood mononuclear cells.
Exome library capture was performed with the Agilent SureSelect Human All Exon V6 followed by sequencing on the Illumina HiSeq 2500. Low-quality raw reads with joint sequences and Phred-scaled single nucleotide polymorphisms (SNPs) quality of < Q20 were removed by using Cutadapt software and SolexaQA software respectively. High-quality clean reads were mapped and aligned to the reference human genome (hg19) by using Burrows-Wheeler Aligner (BWA) [10]. Polymerase chain reaction (PCR) repeats were removed by Picard tool. All raw variants were annotated with Sorting Intolerant from Tolerant (SIFT), PolyPhen-2, Mutation-Taster, and Combined Annotation-Dependent Depletion (CADD). If required, the variants are validated by Sanger sequencing and real-time quantitative PCR (RT-qPCR).

Bioinformatics analysis
Only nonsynonymous single nucleotide variants (SNVs) were considered. According to the 1000 Genomes database, SNVs with the minor allele frequency (MAF) < 0.01 in the general population were considered to be rare, which were potentially signi cant variants. Variants with CADD score > 10 were predicted deleterious variations. Afterwards, candidate variants were considered in twins, or in twins and father, or in twins and mother. Then, we focused on the variants tied to immunologic pathways (www.genecards.org). Protein structure of mutated genes were predicted online (www3.cmbi.umcn.nl/hope/).

Variants likely associated with CVID
Firstly, subtractive analysis of the variant call les between the affected and unaffected family members was carried out using recessive and dominant models according to the law of Mendelian inheritance. The two models respectively revealed 41 and 9 variants but none were predicted to have a deleterious effect on protein function according to SIFT, PolyPhen-2, Mutation-Taster, or CADD.
Then we ltered out 1845 nonsynonymous SNVs from 3401 variants shared by the twins. Among them, 675 rare variants (MAF < 0.01) were screened out in 1000 Genomes database. 398 candidate variants (heterozygous) were predicted to be deleterious based on criteria with CADD score over than 10. But no previously published monogenic CVID variants were identi ed. Systematic assessment of bioinformatical predictions, published literature, gene annotation and the clinical and immunological phenotype of CVID, reduced the list to seven candidate variants have been reported to confer susceptibility to the disease or to originate similar phenotypes to CVID. The bioinformatics analysis process is shown in Fig. 1c.  Table 2).

VDR de ciency
Results of Sanger sequencing identi ed the heterozygous VDR mutation (chr12-48276554: G/T, NM_001017536: exon2: c.71C > A: p.A24D) in the twins and their mother (Fig. 2a). The expression of VDR gene after mutation was signi cantly lower than that of father's wild type (Fig. 2b). The p.24 mutation was located at the DNA-binding domain (DBD) (Fig. 2c). The DBD is organized into two zinc-nucleated modules, which called as vitamin D-responsive elements (VDREs). Mutations in DBD would result in defective DNA binding and the most severe clinical phenotypes of vitamin D resistance. Through online prediction, we found that the mutant amino introduces a charge, which can cause repulsion of ligands or other residues with the same charge. The mutant residue is bigger than the wild-type and might lead to bumps. The hydrophobicity of the mutant residue will be lost, either in the core of the protein or on the surface (Fig. 2d). VDR and Vitamin D play an important role in the innate immune system and modulate Toll-like receptorrelated responses [11]. VDR can affect immune information transmission and B cell activation by affecting calcium ions [12]. Ardeniz [13] found that VDR expression in patients with CVID were lower in the peripheral blood mononuclear cells and hair follicles when compared to the healthy group. Apparently, VDR also affects bone metabolism. We speculate that osteoporosis symptoms in the twins are associated with VDR mutations. Their increased serum ALP may con rm the association.

NHEJ1 de ciency
Heterozygous mutation of NHEJ1 (chr2-220012433: T/C, NM_024782: exon4: c.475A > G, p.M159V) occurred in the twins and their mother (Fig. 2e). The expression of mutant NHEJ1 was signi cantly lower than that of the father's wild type (Fig. 2f). Analysis by the HOPE software suggested that the mutant residue is smaller than the wild-type (Fig. 2g). This will cause a possible loss of external interactions. The mild mutation should not cause misfolding of the protein, but the extra acquired Valine may lead to greater hydrophobicity and may change the interaction between the protein and the corresponding protein (Fig. 2h).
CVID associated with defective DNA double-strand breaks (DNA-DSBs) repair [14]. NHEJ1 mediates the predominant pathway in DSB repair in mammalians, especially during V(D)J recombination. V(D)J recombination defects will block differentiation of T and B cells, then lead to immune de ciency in patients [15]. So, we speculate that the NHEJ1 mutation may be involved in the pathogenesis of the twins.

NOD2 de ciency
NOD2 has been reported to be a possible disease modifying polymorphism in CVID [16]. NOD2 was initially observed for its role in the in ammatory bowel condition Crohn's disease and NOD2-RIP2/NF-κB signaling activation [17]. Watery diarrhea is one of the classic hallmarks of Crohn's disease. NF-κB signaling has a vital role in B and T cell activation and development. Thus, we supposed that NOD2 variant may be related to the CVID twins and the clinical manifestation of diarrhea.

DOCK5 de ciency
As an atypical guanine nucleotide exchange factor, DOCK5 has been extensively studied in cellular functions. Chen [18] has shown that DOCK5 regulates the peripheral B cell differentiation via controlling the CD19-BTK signaling axis as well as actin reorganization through a DOCK5 knockout mouse model. The variant of c.824A > G is novel and has not been reported before. It may be a harmful change through the prediction of PolyPhen-2, Mutation-Taster and CADD.

C3 de ciency
The complement system and Toll-like receptors (TLRs) are key elements of innate defense mechanisms [19]. B lymphocytes express both TLRs and complement receptors (CRs) [20]. Simultaneous or sequential engagement of these structures expressed either on the cell membrane or intracellularly, may fundamentally alter and ne tune activation, antibody and cytokine production of B cells. Activation products of the complement system, particularly C3-derived fragments, play an important role in the regulation of B cell responses by binding to their corresponding receptors [21][22][23]. In our case, C3 variants (c.2851C > T and c.1940C > T) may be harmful changes through the prediction of SIFT, PolyPhen-2 and CADD.

Acquired pure red cell aplasia
CVID is prone to autoimmune thrombocytopenia, hemolytic anemia and other hematological disorders [24]. As for immunosuppressive agent, cyclosporine shows effective to granulomatous diseases [25] and non-infection diarrhea in CVID patients by suppressing abnormal activation of B cells and T cells [26]. Cyclosporine also has a rapid control effect on our proband regarded as PRCA associated CVID. After 5month's treatment with cyclosporine, the patient was relieved. This is different from the case Sklarz T. [27] reported that a female was diagnosed with CVID at her 12 years' old. When she was 48 years' old, she was notable for a low reticulocyte count and a hypocellular marrow with an absence of erythroid precursors, consistent with acquired red cell aplasia. However, she was treated with cyclosporine without response and eventually progressed to aplastic anemia at her 50's. The pathogenesis of PRCA happened in CVID is unrevealed, but the phenomenon implies that lack of antibody responses may lead to aberrant cytotoxic T lymphocyte (CTL) responses with cross-reactivity directed to red cell precursors [28].

Conclusion And Prospect
With the success of new sequencing technologies, and in particular of WES in unraveling the molecular mechanisms of many rare syndromes, rare diseases such as CVID that do not completely t with a Mendelian model represent a new challenge for medical genomics. Monozygotic twins are commonly considered to have a similar inherent genetic background and provide a powerful model to dissect the relative contributions of heritable and non-heritable variables to the establishment of clinical and immunological pro les. Silva SL [9] reported a pair of monozygotic twins with CVID have strikingly similar clinical and immune pro le associated with a polygenic burden, despite exposure to different living conditions. However, the clinical manifestations of monozygotic twins in our study were not concordant. Only the elder brother developed pure red aplastic anemia. Moreover, variant genes related to CVID susceptibility mainly inherited from their mother, but the mother only showed decreased bone density. It is suggested that these variants have an autosomal dominant inheritance with an incomplete penetrance. Li [29] carried out the WES on three sporadic CVID patients and their offspring and provided similar evidence. Some monogenic disease-causing mutations also appeared in their unaffected offspring.
In conclusion, we described the clinical phenotypes of monozygotic twins of CVID and tried to identify genetic defects using WES. It failed to identify a monogenic cause for CVID in the family, but we screened VDR, NHEJ1, DOCK5, NOD2 and C3 as candidate pathogenic genes. We speculated the potential pathogenic mechanism for combinatorial effects in CVID is inferred from their roles in T and B cell signal pathways activation (Shown in Fig. 2i). We supposed that the onset of twins may be caused by multiple epistatic interactions [30] and cumulative effects, which means a polygenic pattern. Our study has some limitations. Firstly, the twins are unmarried and have no children for genetic testing. Secondly, the functional effects of these candidate mutants are not veri ed but have provided additional information for clinical management. Thirdly, it should be mentioned that other variants like non-coding variants or deep intronic variants resulting in splicing defects or epigenetics or CNV analysis may be the possibility because these variants cannot be detected in this WES study.
Since CVID forms a heterogeneous group of phenotypes and genotypes, genetic investigation is still a great challenge. A combination of clinical and genetic diagnosis can be more extensively utilized in the clinical practice of CVID. Disease-related gene variations need to be found and con rmed in more patient populations and further studies, which will help to profoundly improve our understanding of CVID. The nucleated erythrocyte was only 1.5% (<5%) and early erythroblast was observable. c Filtering strategies for candidate SNVs in the study. The number of genes ltered was within the parentheses.  The picture shows the proteins encoded by the selected candidate genes involved in the development and maturation of B cells, as well as the role and interaction of T cell and B cell signal transduction. During B cell development, pre-B cells undergo V(D)J rearrangement, which triggers RAG-induced DNA doublestrand damage repair (RAG-DSBs). Similarly, activated B cells undergo class-switching reorganization. This process triggers AID-induced DNA Double-strand damage repair (AID-DSBs). In both of the above processes, NHEJ1 is a key protein involved in DNA double-strand damage repair. Abnormal activation of the nuclear factor-κB (NF-κB) has been reported to be related to the pathogenesis of CVID. VDR protein is a membrane protein that regulates calcium ions positively after being activated, which further activate NF-κB and the nuclear factor of activated T cells transcription factor (NF-AT) signaling pathways. NOD2 protein also can regulate NF-κB signaling pathway. DOCK5 can play an immunoregulatory role by stimulating the BCR signaling pathway. As for the complement C3 protein, its cleavage product C3b can be combined with the BCR costimulatory factor CD21 to activate the BCR signaling pathway, and ultimately promote NF-κB signaling pathway to regulate the immune response.