A novel hemizygous CD40L mutation of X-linked hyper IgM syndromes and compound heterozygous DOCK8 mutations of Hyper IgE syndromes in two Chinese families

Background X-linked hyper IgM syndromes (X-HIGM) and autosomal recessive hyper IgE syndromes (HIES) are rare primary immunode�ciency diseases, characterized by recurrent infections due to the impairment in immune system. This study was aimed to investigate genotype-phenotype association and reveal the novel likely pathogenic mutations in CD40L and DOCK8 responsible to patients with X-HIGM and HIES respectively. Methods Whole exome sequencing (WES) and Sanger sequencing were performed to identify and verify the likely pathogenic mutations in the two families. Results


Introduction
Primary immunode ciency diseases (PIDs) are a heterogeneous group of inherited disorders impairing both innate and adaptive immunities of affected patients [1], and patients are susceptible to develop recurrent infectious diseases, autoimmune diseases and malignancies, which leading to signi cant morbidity and mortality.The prevalence of PIDs varies among different populations with approximately 10:100 000 live births annually worldwide [2] and 1:1200 live births in US [3].More than 300 PIDs have been identi ed until now [4], and most of them was caused by single gene variants [5].Immunode ciencies can be generally classi ed into two groups according to disrupted component in immune system: 1) disorders of adaptive immunity with T lymphocytes, B lymphocytes or combined de ciency; 2) disorders of innate immunity with phagocyte and complement de ciency [6].Particularly, PIDs with B lymphocytes de ciency is the most common type.The basic treatments for patients include immunoglobulin supportive therapy and anti-in ammatory treatment.
Hyper-IgM (HIGM) syndrome as a type of PIDs, initially described by Rosen in 1961 [7], is characterized by normal or elevated serum levels of IgM but low or absent serum levels of IgG, IgA and IgE [8], which leads to development of recurrent infections, including upper and lower respiratory tract infection, diarrhea, and various opportunistic infections [9][10][11][12], autoimmune de ciencies, lymphoid hyperplasia, and malignant tumors [13,14], resulting in high mortality of young patients [15].X-linked recessive mode of HIGM (XHIGM, OMIM:308230), the most common format of HIGM, usually results from mutations in CD40 ligand (CD40L) located at Xq26.3-Xq27.1 and composed of 4 introns and 5 exons spanning 12214bp [16][17][18].CD40L expressed on activated T cells as the type II membrane glycoprotein interacts with CD40 receptor expressed on antigen-presenting cells including B lymphocytes and dendritic cells [19], to further activate T cells and promote the development and maturation of B lymphocytes [20], and regulate immunoglobulin isotype switching in B lymphocytes from IgM to IgG, IgA and IgE [21].IgM acts as the rst antibody against pathogenic microorganisms [22], the failure of isotype switching causing the development of HIGM.
Hyper-immunoglobulin E syndrome (HIES), also known as Job's syndrome, was rstly reported as a primary immunode ciency disease in two patients presenting recurrent pulmonary infections and cold staphylococcal abscesses [23].The clinical manifestations of HIES include recurrent staphylococcal "cold" skin and pulmonary abscesses, increased serum level of IgE and eosinophilia, eczematoid dermatitis and de ciency in T cells functions [24,25].HIES was typically classi ed into autosomal dominant HIES (AD-HIES) and autosomal recessive HIES (AR-HIES) according to the inheritance format [26].Previous studies have indicated that AR-HIES was mainly caused by mutations in the dedicator of cytokinesis-8 (DOCK8) mapped on chromosome 9p24 containing 47 or 48 exons spanning 190kb [27].DOCK8 defect impaired both CD4 + and CD8 + T cells activation and responses, leading to the de ciency in cellular and humoral immunity, thus patients were susceptible to microorganism infection and presented the severe clinical symptoms [28].
As the clinical manifestations and laboratory ndings are insu cient to make de nitive diagnosis of PIDs due to the complexity, the turnaround time was largely shortened and the diagnosis was accelerated which enables patients to receive timely treatment and facilitates genomics research with the rapid development of next-generation sequencing (NGS) [29].In this study, we described two cases of PIDs, including a X-HIGM case caused by a novel hemizygous mutation in CD40L and an AR-HIES case by the compound heterozygous mutation in DOCK8, and constructed the genetic and phenotype relationship of PIDs, which broaden our knowledge to pathogenic variant spectrum of X-HIGM and HIES, thus promote diagnostic accuracy and facilitate timely treatment.

Patient
The selected probands were from two Chinese Han families in Shandong province.The rst 4-month-old proband with was admitted to the hospital with the symptoms of recurrent shortness of breath, hoarseness and cough for a month.He was the rst child in a non-consanguineous healthy family and delivered by cesarean section at full-term.The second proband was admitted to hospital two hours after delivered by cesarean section at 38 weeks with the symptoms of perioral cyanosis, shortness of breath, groaning, and not crying.They were diagnosed based on clinical manifestations and laboratory examinations.The study was approved by Medical Ethics Committee of The A liated Hospital of Qingdao University.WES analysis and Sanger sequencing validation were performed after blood samples collected from patients and their families.

Methods
Whole exome sequencing(WES) At least 2 ml peripheral blood was obtained from two patients and their families.The genomic DNA was extracted from blood using a Qiagen DNA extraction kit following the manufacturer's protocol and was quanti ed by spectrophotometer.Subsequently, the quanti ed DNA samples were randomly fragmented into 180-250 bp using a Covaris S220 sonicator (Covaris, Inc.).DNA fragments were end repaired by adding T4 DNA polymerase and polynucleotide kinase (PNK; Vazyme Biotech Co, Ltd), and polymerase chain reaction (PCR) ampli cation was performed to enrich DNA fragments, thus the sequencing libraries were constructed.In addition, an Agilent 64M liquid phase chip capture system was conducted to capture the exon regions and PCR was subsequently performed to enrich the exon DNA libraries.Finally, the quali ed libraries were sequenced on Ιllumina NextSeq platform for paired-end 150bp reads.All exon regions and the clipping regions were sequenced by WES analysis for mutation detection.

Sanger sequencing validation
The detected variants of CD40L and DOCK8 were further veri ed by Sanger sequencing.Ampli cation was performed by PCR and the primers were designed using Primer Premier version 5.0 software: primer sequence of CD40L, forward-TGATGCCGTGGAAATGAATG, reverse-TTTTGCCTAGTGGTAGCTGCATAT, primer sequence of DOCK8 (c.1546C > G), forward-CTCTAATCCATTCCTTCTCATTTTACAAT, reverse-AAACTCCTGAGCTCAAGCAATCC, primer sequence of DOCK8 (c.5355 + 6 C > T, splicing), forward-TGTTTGGACAATGACCTCTGGTT, reverse-ATAACAACAAAAGGAATCCCATGAA.The steps of PCR reaction were as follow: 95 ℃ for 5 min followed by 40

Clinical manifestations
The rst 4-month-old male proband (P1) with X-HIMG (Fig. 1A During hospitalization, the proband received Beta-lactam antibiotics including cephalosporin antibiotics, meropenem, and Piperacillin Sodium and Tazobactam Sodium against bacterial infections, uconazole against fungal infections, erythromycin against atypical pathogens, and ganciclovir against HCMV infection.Other supportive therapies included mask oxygen inhalation, methylprednisolone for antiin ammatory treatment and intravenous immunoglobulin (IVIg).The patient was discharged from the hospital when he got better.
The second male proband (P2) of HIES (Fig. 1B) was hospitalized two hours after delivered by cesarean section at 38 weeks with a birth weight of 3200g.Apgar score was 7 scores (Respiration, Appearance, Activity, -1) at 1 minute without intrauterine fetal anoxia and premature rupture of membrane, 8 and 9 scores at 5 and 10 min with positive-pressure ventilation.The patient presented perioral cyanosis, shortness of breath with 55 breaths/min, groaning, and not crying.Physical examinations: body temperature 36.4 degrees, heart rate 110 beats/min, blood pressure 63/30 mmHg, and poor responses.Thick respiratory and wet rales were audible in both lungs and the fetal SpO 2 was about 85 percent without oxygen inhalation.The cranial MR and chest CT examination separately indicated subarachnoid hemorrhage and pneumonia.In addition, the cornea of his both eyes were milky white and the pupils were invisible.The following ophthalmology ultrasonography described obvious corneal edema and thickening, narrow atrial angle in all directions and short axial diameter.The patient was diagnosed with congenital microphthalmia, congenital leucoma, and secondary glaucoma by ophthalmologist.He soon developed convulsion with regular shaking and increased muscle tension and was given phenobarbital for sedation and spasmolysis.His condition deteriorated rapidly with the treatment of oxygen inhalation, intravenous IVIg and cefotaxime anti-infections, manifesting as poor response, irregular breath and pale skin.Blood gas analysis indicated metabolic acidosis and hyperkalemia.Echocardiography showed a decreased left ventricular systolic function (the ejection fraction was 28%), which indicated a heart failure of the patient.His parents gave up continuing treatment and signed for discharge due to his severe symptoms and little hope of survival.The proband was the second child in this non-consanguineous healthy family, in which the rst child was a female infant and died of asphyxia neonatorum at 7 days after delivery.However, WES analysis was not performed in the rst child.

Genetic analysis
WES analysis revealed a novel hemizygous mutation in CD40L (c.257delA or deletion of adenine at nucleotide position 257) in P1.This frame shift mutation resulted in the substitution of glycine for glutamic acid at 86 codon of the protein causing the early termination of translation at downstream codon 9 (p.E86Gfs*9).Sanger sequencing was then utilized to verify the detected variant in CD40L by WES analysis (Fig. 2A) and showed that the hemizygous c.257delA mutation was inherited from his mother.Besides, this deletion mutation was not found in 100 irrelevant healthy people and was likely responsible for the X-HIGM phenotype.
We identi ed the compound heterozygous mutations in DOCK8, one in exon ).The compound heterozygous mutations were independently inherited from his patents, both of which were not found in 100 irrelevant healthy people.His father was heterozygote of c.1546C > G mutation and his mother was heterozygote of c.5355 + 6C > T (splicing) mutation (Fig. 2B, C).Therefore, the co-segregation analysis in this family indicated that the compound heterozygous mutations were likely pathogenic in HIES phenotype.

Bioinformatic analysis of CD40L and DOCK8 mutations
The CD40L and protein sequences of different species, including Homo sapiens, Rattus norvegicus, Mus musculus, Canis lupus familiaris, Felis catus, Sus scrofa and Macaca mulatta, were obtained from the National Center for Biotechnology Information (NCBI) website.Multiple sequence alignment among these species were compared using DNAMAN software, demonstrating that the variant -p.E86-was located at a highly conserved sequence of CD40L (Fig. 3A).The DOCK8 and protein sequences were searched in Homo sapiens, Mus musculus species, Canis lupus familiaris, Macaca mulatta and Rattus norvegicus.
The result indicated that the variant p.P516A was located at a highly conserved sequence of DOCK8 (Fig. 3B).

Discussion
In the present study, we identi ed a novel hemizygous mutation (c.257delA) of CD40L in P1 with X-HIGM and the compound heterozygous mutations (c.1546C > G/ c.5355 + 6C > T) of DOCK8 in P2 with HIES by WES analysis together with Sanger sequencing validation, expanding our knowledge to the pathogenic variants of CD40L and DOCK8.So far, more than 130 variants have been identi ed in CD40L.In previous study, we reported a novel deletion variant (c.714delT in exon 5, p.F238Lfs*4) of CD40L in a Chinese family affected by X-HIGM [30].The compound heterozygous mutations including c.1546C > G and splice-site mutation c.5355 + 6C > T in DOCK8 were separately inherited from his parents.Bioinformatic analysis and co-segregation analysis indicated that the c.257delA mutation in CD40L was associated with the phonotype of HIGM and the compound heterozygous mutations in DOCK8 resulted in the phonotype of HIES.However, further functional veri cation is needed to make de nitive conclusion.
As a group of rare immunode ciency diseases, X-HIGM is attributed to the impairment of immunoglobulin isotype switching, which results from defects in CD40 ligand/CD40 signaling pathway in lymphocytes caused by pathogenic mutations of CD40L.The patients present the de ciency in humoral and cellular immunity and are susceptible to recurrent infections including intracellular, opportunistic and extracellular pathogens [31].The clinical features of X-HIGM are variable and complex.Winkelstein et al. involved 79 patients with X-HIGM and described their clinical manifestations.The most prominent clinical manifestation was increased susceptibility to infection of P.carinii, viral and fungal organisms including cytomegalovirus, adenovirus, herpes simplex virus type I and so on.These patients presented pneumonia (81%), upper respiratory infections (49%), protracted or recurrent diarrhea (34%), central nervous system (CNS) infections (14%), sepsis (13%) caused by pneumococcus and Pseudomonas, hepatitis (9%), sclerosing cholangitis caused by Cryptosporidium and neutropenia [32].We previously described a case admitted to hospital twice for the tuberculous in ammation and HCMV infection [30].Recently, a study from Italy described a patient who developed cryptococcal meningoencephalitis after receiving regular antibiotic treatment and immunoglobulin supplementation [33].Gallagher et al. described a 5-month-old male patient with c.608G > C mutation in CD40L presenting respiratory failure due to diffuse pulmonary alveolar proteinosis (PAP) [34].A European group reported the outcomes in 56 patients diagnosed with X-HIGM that 23.2% of patients died between the age of 9 month and 23 years old mostly due to the infections or liver failure [35].Long-term survival outcome of X-HIGM was poor with the treatment of antiinfection and supportive therapy.Hematopoietic stem cell transplantation was suggested to be the only curative treatment for these patients [36].Wang et al collected clinical manifestations of 20 Chinese patients with X-HIGM and showed that the complex symptoms included recurrent sinopulmonary infections (18 patients, 90%), neutropenia (14 patients, 70%), oral ulcer (13 patients, 70%) and protracted diarrhea (13 patients, 65%) [18].The main clinical features in China are different from western countries, indicating clinical manifestation ethnic differences.Recently, anti-phospholipid syndrome was rstly reported as a novel complication in a Vietnam patient with X-HIGM [37].In this study, the rst 4-month-old male proband with X-HIGM presented severe pneumonia by HCMV infection and hypogammaglobulinemia.However, He had no neurological symptoms and no presentation of diarrhea and liver dysfunction.
Novel homozygous or compound heterozygous mutations in DOCK8 were found to be responsible for a large proportion of AR-HIES by encoding defective DOCK8 protein, independently reported by Engelhardt and Zhang in 2009 [28,38].This kind of primary immunode ciency disorder has increased serum level of IgE and eosinophilia.DOCK8 protein, a member of DOCK180 superfamily, was involved in organization of actin lament system by interacting with the Rho GTPases which participate in critical cell processes including cell migration, cell survival and cell transcriptional regulation [27].[40].In our study, the second proband affected by HIES presented neonatal pneumonia, severe asphyxia neonatorum and cerebral manifestation including convulsion and subarachnoid hemorrhage.Except the symptoms mentioned above, this patient was diagnosed with congenital microphthalmia, congenital leucoma and secondary glaucoma.No association between HIES and congenital malformation of eyes and no correlation between congenital microphthalmia and DOCK8 mutations were reported until now after searching the relevant studies previously published.Congenital microphthalmia was associated with the mutations of CRYBB3, FOXE3 and PAX6, none of which was detected by WES analysis in this patient [41][42][43].Therefore, we recommended the couple to seek genetic counseling positively before their next pregnancy.
As primary immunode ciency disorders, X-HIGM and HIES present heterogeneous manifestations mainly characterized by recurrent multiple site infections to opportunistic pathogens with the impairment in humoral and cellular immunity, increasing the di culty of clinical diagnosis.Long-term outcome with supportive therapy and anti-infection was poor especially in young patients.We detected the defective genes in two pedigrees applying WES analysis and Sanger sequencing validation.As a result, the rst proband was discovered to have a novel pathogenic deletion at exon 2 of CD40L and the second proband carried the compound heterozygous mutations in DOCK8, including a mutation at exon 14 and a splicing mutation at exon 41.In conclusion, the above ndings broaden our knowledge to the likely pathogenic variant spectrum of X-HIMG and HIES, which is helpful for promoting diagnostic accuracy, facilitating timely treatment and improving long-term survival of affected patients.
cycles of ampli cation, denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, and elongation at 72 ℃ for 30 s.The ampli ed PCR products were puri ed and analyzed by ABI 3730XL sequencer (Applied Biosystems 3730XL).The potential pathogenic mutations were compared to the reference sequence on National Center Biotechnology Information (NCBI) website.Bioinformatics software was utilized to predict the candidate mutation of CD40L and DOCK8.The c.1546C > G variant was predicted by software including Polymorphism Phenotyping v2 (http://genetics.bwh.harvard.edu/pph2/),PROVEAN ( http: //provean.jcvi.org/index.php) and MutationTaster (http: //www.mutationtaster.org).The c.5355 + 6C > T(splicing) variant was predicted by MaxEntScan (http: //genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html),Spliceman (http:// 31E + 02 copies/mL, the bronchoalveolar lavage uid of HCMV-DNA 2.25E + 05 copies/mL.Mycobacterium tuberculosis test was negative.Based on the clinical and laboratory ndings, the preliminary diagnoses were severe pneumonia, acute respiratory failure and hypogammaglobulinemia. ) was the rst child in a nonconsanguineous healthy family and delivered by cesarean section at full-term with a birth weight of 3800g.Apgar score for him was 10 scores at 1, 5 and 10 min after birth.This patient presented recurrent shortness of breath, hoarseness and cough for a month without apparent inducement.He was admitted to hospital on March 10, 2019, after ineffective treatment with Amoxicillin and Clavulanate Potassium for 4 days.Physical examinations: shortness of breath with 38 breaths/min, dyspnea with positive triconcave sign, slightly perioral cyanosis, hoarseness, cough, body temperature 37.1 degrees, heart rate 152 beats/min, and blood pressure 84/55 mmHg.The oxygen saturation rate (SpO 2 ) was about 78-85 percent without oxygen inhalation.During the emergency observation, the patient developed increased cyanosis and was given mask oxygen inhalation mixed with nebulized budesonide, intravenous infusion of erythromycin and methylprednisolone.The peripheral blood examination showed a high level of white blood cell (18.32*10 9 /L, reference value: 5-12*10 9 /L), and the chest CT (Computed Tomography, CT) indicated pneumonia.Subsequent laboratory examinations: the serum IgG < 1.360 g/L (Reference value:1.39-9.34g/L), IgA 0.04 g/L (Reference value: 0.03-0.78g/L), IgE 16.70 IU/mL (Reference value: 0-60 IU/mL), IgM 0.23 g/L (Reference value: 0.04-1.2g/L); the serum Cytomegalovirus DNA (HCMV-DNA) test 4.
[39]patients were characterized by recurrent viral and bacterial infections mainly including herpes simplex virus, human papillomavirus and Staphylococcus aureus infection, molluscum contagiosum[39], candidiasis [28], atopic eczema, recurrent upper and lower respiratory tract infections, extensive anaphylaxis, otitis media, and sinusitis[38].Notably, Engelhardt et al described rare cerebral manifestations including CNS vasculitis, brain infarction, meningitis and multifocal leukoencephalopathy [28].Yang et al described a 7year-old girl diagnosed with HIES with two compound heterozygous DOCK8 mutations, presenting neurological symptoms including abnormal gait, high muscle tone and facial paralysis