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RESEARCH ARTICLE

Whole-exome sequencing identified a homozygous novel RAG1 mutation in a child with omenn syndrome

Wendi Wanga, Jian Wanga, Jingjing Wanga, Jingting Liua, Jianying Peia, Wanyi Lia, Yanxia Wanga, Santasree Banerjeeb, Ruifeng Xua*, Zhaoyan Menga*, Bin Yia*

aGansu Provincial Maternity and Child-Care Hospital, 143, North Street, Qilihe District, Lanzhou, Gansu Province 730050, China

bDepartment of Genetics, College of Basic Medical Sciences, Jilin University, Changchun, China

Abstract

Introduction and objectives: Omenn syndrome (OS) is a very rare type of severe combined immunodeficiencies manifested with erythroderma, eosinophilia, hepatosplenomegaly, lymph-adenopathy, and elevated level of serum IgE. OS is inherited with an autosomal recessive mode of inheritance. Germline mutations in the human RAG1 gene cause OS.

Materials and methods: In this study, we investigated a 2-month-old boy with cough, mild anaemia, pneumonia, immunodeficiency, repeated infection, feeding difficulties, hepatomegaly, growth retardation, and heart failure. Parents of the proband were phenotypically normal.

Results: Karyotype analysis and chromosomal microarray analysis found no chromosomal structural abnormalities (46, XY) and no pathogenic copy number variations (CNVs) in the proband. Whole-exome sequencing identified a novel homozygous single nucleotide deletion (c.2662delC) in exon 2 of the RAG1 gene in the proband. Sanger sequencing confirmed that both the proband parents were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters and one elder brother of the proband and in the 100 ethnically matched normal healthy individuals. This novel homozygous deletion (c.2662delC) leads to the frameshift, which finally results in the formation of the truncated protein (p.Leu888Phefs*3) V(D)J recombination-activating protein 1 with 890 amino acids compared with the wildtype V(D)J recombination-activating protein 1 of 1043 amino acids. Hence, it is a loss-of-function variant.

Conclusions: Our present study expands the mutational spectrum of the RAG1 gene associated with OS. We also strongly suggested the importance of whole-exome sequencing for the genetic screening of patients with OS.

Key words: frameshift mutation, immune responses, omenn syndrome, RAG1 gene, SCID

*Corresponding authors: Dr. Ruifeng Xu, Gansu Provincial Maternity and Child-Care Hospital, 143 North Street Qilihe District, Lanzhou, Gansu Province 730050, China. Email address: [email protected]; Dr. Zhaoyan Meng, Gansu Provincial Maternity and Child-Care Hospital, 143 North Street Qilihe District, Lanzhou, Gansu Province 730050, China. Email address: [email protected]; Dr. Bin Yi, Gansu Provincial Maternity and Child-Care Hospital, 143 North Street Qilihe District, Lanzhou, Gansu Province 730050, China. Email address: [email protected]

The authors contributed equally to the work.

Received 24 October 2021; Accepted 13 June 2022; Available online 1 November 2022

DOI: 10.15586/aei.v50i6.529

Copyright: Wang W, et al.
License: This open access article is licensed under Creative Commons Attribution 4.0 International (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

Introduction

Human severe combined immunodeficiency (SCID) is a large group of genetic disorders with extreme genotypic and phenotypic heterogeneity.1 SCID is usually characterized by severe, recurrent, and potentially lethal infections that occur soon after birth or early in infancy. Patients with SCID usually manifest with failure to thrive and lymphopenia of T lymphocytes.2 SCID patients also presented with a deficiency of antibodies and complete loss or marked abnormalities in cell-mediated immunity.3 SCID patients were classified into two types based on the presence (B+ SCID) or absence (B SCID) of B cells in the peripheral blood.4,5

Omenn syndrome (OS) is a very rare type of inherited SCID presented with several early postnatal or infancy onset manifestations, i.e., diffuse exfoliative erythroderma, lymphadenopathy, hepatosplenomegaly protracted diarrhoea, alopecia, failure to thrive, eosinophilia, elevated level of serum immunoglobulin E (IgE) with typical recurrent severe life-threatening infections.6 Laboratory investigations of SCID patients usually showed eosinophilia and T-cell lymphocytosis.7 According to immunological phenotypes, OS is classified into two types, namely T+BNK+ and T−BNK+.8 To date, OS could only be treated by bone marrow transplantation or cord blood stem cell transplantation.8

Germline mutations in recombination-activating genes 1 (RAG1) cause the majority of OS.9 RAG1 gene is located at the short arm of chromosome 11 (11p12). RAG1 gene encodes a lymphoid-specific complex of enzymes named V(D)J recombination-activating protein 1. This protein plays a significant role in initiating the V(D)J recombination process.10 During the development of T-cell and B-cell receptors, V(D)J recombination-activating protein 1 is involved in the process of rearrangement of the variable (V), diversity (D), and joining (J) segments. Both RAG1 and RAG2 proteins combined and cleaved the recombination signal sequences (RSSs), variable (V), diversity (D), and joining (J) gene exons and initiating the recombination process of variable (V), diversity (D), and joining (J) segments. This process of somatic recombination leads to the formation of diverse T-cell and B-cell repertoires.11 Germline mutations in RAG1 genes causes complete loss or reduced recombination V(D) J followed by the loss of the development of both B and T cell, which finally results in the deficiency of circulating B cells and non-functional oligoclonal T cells, leads to severe combined immunodeficiency (SCID).12,13 In addition, RAG1 hypomorphic mutations lead to the formation of partially functional RAG1 protein followed by the partial development of B and T lymphocytes which in turn causes phenotypic heterogeneity and a spectrum of severe immunodeficiencies.14,15 According to the different levels of RAG1 expression, patients with OS showed a spectrum of immunological phenotypes and diverse clinical manifestations.16

Here, we presented the case of a 2-month-old patient clinically diagnosed with OS. Karyotype and chromosomal microarray analyses found no chromosomal structural abnormality in the proband (46, XY) and did not identify any pathogenic copy number variations (CNVs) in the chromosomes of the proband. Whole-exome sequencing (WES) identified a novel homozygous deletion (c.2662delC) in exon 2 of the RAG1 in the proband. Sanger sequencing confirmed that both the proband parents were carrying this variant in a heterozygous state. This variant leads to the truncated RAG1 (p.Leu888Phefs*3) protein with 890 amino acids compared with the wildtype RAG1 protein of 1043 amino acids. Our present study expands the mutational spectrum of the RAG1 gene associated with OS. We also strongly suggested the application of WES for accurate, timely, and cost-effective screening for patients with OS.

Materials and Methods

Patient and clinical samples

A Hui Chinese infant with OS was enrolled in our hospital (Gansu Provincial Maternity and Child-Care Hospital, Lanzhou, Gansu Province, People’s Republic of China). Blood samples were collected for analysis. The study was approved by the ethics committee of our hospital (Gansu Provincial Maternity and Child-Care Hospital, Lanzhou, Gansu Province, People’s Republic of China), following the Declaration of Helsinki guidelines. We obtained written informed consent from all of the participants of this study.

Karyotype and chromosomal microarray analyses

We performed standard G-banding karyotyping to analyse the structure of all chromosomes in the proband. Next, to confirm the presence of copy number variations (CNV) in the proband, chromosome microarray analysis was performed using a CytoScan HD array (Affymetrix), according to the manufacturer’s protocols (Affymetrix). Chromosome Analysis Suite software version 1.2.2 was used for analysing the data. The reporting threshold of copy number was set at 10 kb with the marker count ≥50.17

Whole-exome sequencing

Blood samples were collected and genomic DNA was extracted from proband using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Proband’s genomic DNA was subjected to WES. Agilent SureSelect version 6 (Agilent Technologies, Santa Clara, CA) was used for capturing sequences. Then, the enriched library was sequenced on an Illumina HighSeq4000. Next, WES reads were aligned to the GRCh37.p.10 by using Burrows–Wheeler Aligner software (version 0.59). After that, GATK IndelRealigner was used for local realignment of the Burrows–Wheeler aligned reads. Then, the base quality recalibration of the Burrows–Wheeler aligned reads was performed by using the GATK Base Recalibrator. Next, the identification of single-nucleotide variants (SNV) and insertions or deletions (indel) was done by the GATK Unified Genotyper. Then, variants were annotated with the Consensus Coding Sequences Database (20130630) at the National Center for Biotechnology Information. Illumina pipeline was used for image analysis and base calling. Indexed primers were used for data fidelity surveillance. In order to align the clean sequencing reads with the human reference genome (hg19), SOAP aligner (soap2.21) software was used. Then, to assemble the consensus sequence and call genotypes in target regions, we used SOAPsnp (v1.05) software.

Bioinformatics analysis

Identified variants by WES were selected for data interpretation with minor allele frequency <0.01 in dbSNP, Hapmap, 1000 Genomes Project, and our in-house database for 5,000 Chinese. Data analysis was performed based on the variant interpretation guidelines of the American College of Medical Genetics and Genomics (ACMG).18 Deleterious variations were selected and performed their segregation analysis among the family members. The variants’ function and association with the disease phenotype was done based on the information from OMIM database and previously published literature.

Sanger sequencing

To validate the identified variants by WES, we performed Sanger sequencing. Designing primer pairs for the candidate loci have been done based on the reference genomic sequences of the Human Genome from GenBank in NCBI. Primer pairs were synthesized by Invitrogen, Shanghai, China. Polymerase chain reaction (PCR) was performed with an ABI 9700 Thermal Cycler. Next, directly sequenced the PCR products by an ABI PRISM 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). Analysis of sequencing data has been done by DNASTAR SeqMan (DNASTAR, Madison, Wisconsin, USA).

WES identified the novel homozygous variant, which was validated by Sanger sequencing using the following primers: F1 5'-GGCGCGGGGGTCTCGCGGCG-3', R1 5'-GGCGGCGAATTCTATAGCG-3'. The reference sequence NM_000448.2 of RAG1 was used.

In silico analysis

The variant identified in the proband by WES was analysed by Mutation Taster (http://mutationtaster.org/).19

Data availability

All data used for the analyses in this report supporting the findings of this study are available on reasonable request to the corresponding author.

Results

Human subjects

We investigated a proband of a nonconsanguineous Hui Chinese family. He was the fourth child in his family. He was born after full-term delivery, weighing 3.3 kg and showed normal breast-feeding after birth. The child has two elder sisters and one elder brother who are in good health. No history of genetic and metabolic diseases was found in his family Figure 1.

Figure 1 Pedigree of the Chinese family with OS. Squares and circles denoted males and females respectively. Individuals labelled with a solidus were deceased. Roman numerals indicate generations. Arrow indicates the proband (II-4).

At the age of one month, the child developed coughing, 2–3 sounds/burst, less sputum without wheezing, fever, and hoarseness. He was first admitted to a local hospital and given intravenous infusion (specifically unknown) for 10 days. After that, the proband has been suffering from frequent coughing with bruising around the mouth. He was then admitted to our hospital (Gansu Provincial Maternity and Child-Care Hospital) for intravenous infusion (specifically unknown), and his cough was relieved. Then, he was again admitted to our hospital with coughing accompanied by occasional bruising around the mouth and changes in stool characteristics. However, we have not found the obvious cause of recurrence of frequent coughing in this proband. There was no fever, no breathing difficulties or other symptoms manifested in the proband.

Physical examination revealed that his body temperature was 36.9°C, pulse rate was 170 beats/min, breath rate was 84/min, blood pressure was 87/47 mmHg and body weight was 5.28 kg with 90% SpO2.

The proband clinically manifested normal development, malnutrition, drowsiness, poor mental response and normal skin colour with decreased elasticity. The proband was characterized by dry lips and red throat, and the pharynx was slightly congested with copious white secretions, which could be seen on both sides of the oral mucosa and the isthmus, easy to peel and easy to exfoliate. The pro-band was identified with normal head size with Bregma Depression size of 3.0 × 3.0 cm2. No visual or hearing impairments were found in the proband. The proband presented with thick breath sounds in both lungs with normal breathing movements, strong heart sounds with a heart rate of 170 beats/min, normal rhythm and no murmurs. Normal abdominal appearance with a soft belly, 4.5 cm below the liver ribs palpable, and active bowel sounds were reported. The proband’s gallbladder, spleen and kidneys were normal. The proband’s lymph nodes were normal, and superficial lymph nodes were present throughout the body without any swelling. We identified no abnormal vascular signs in the peripheral blood vessels and no neuro-muscular abnormalities in the proband. During the course of the illness, the proband’s mental state was slightly worse. The proband was manifested with poor appetite but without vomiting and diarrhoea.

In the proband, we found that the colour Doppler of two-dimensional and M-type Doppler measurements (mm) was AO 9 LA 14 IVS 3 LV 20/12 LVPW 3 RV 9 MPA 11 LPA 5 RPA 5 EF 70 (%) FS 40 (%) and Spectrum Doppler ultrasound (cm/s) was MV 85 TV 76 AV 102 PV 98.

The inner diameter of each chamber of the heart was normal. The interventricular septum was not thick. No echo of the arterial catheter was detected, and each valve’s shape and opening and closing were normal. Left ventricular ejection fraction and short-axis contraction rate was in normal range. No abnormal blood flow signal and frequency spectrum were detected.

There was no obvious abnormality in the structure of the heart and blood flow. The left and right ventricular systolic and diastolic functions were normal. There was no obvious abnormality in pulmonary artery pressure.

The chest CT found that the chest was symmetrical with a centrally located trachea. The texture of the two lungs was increased, and the upper field mass flaky high-density shadow was visible in the inner area of the two lungs. The right edge was blurred. The diaphragm muscles were smooth on both sides, and the bilateral costal diaphragm angle was sharp. The proband was clinically diagnosed with “bronchial pneumonia” (Figure 2).

Figure 2 Chest CT examination. (A) The texture of the two lungs increased, with blurred edges. On the right lung, mass-shaped high-density shadows, and bronchial inflation shadows. Severe pneumonia in both lungs, the part of the right lung is actually changed. (B) The texture of the two lungs increased, and the flake high-density shadow and the right lung upper field mass flaky high-density shadow were visible in the inner area of the two lung fields, and the density was lighter. Bipulmonary pneumonia has absorbed and improved better than before. (C) The end of the endotracheal tube is at the level of the lower edge of T1. The double pneumonia is heavier than before. The end of the endotracheal tube is at the level of the lower edge of T1. (D) The tip of the endotracheal tube is at the level of the T1 vertebral body. The double pneumonia is heavier than before, the tip of the endotracheal tube is at the level of the T1 vertebral body. (E) The tip of the endotracheal tube is at the level of the T2 vertebral body. Severe pneumonia of the two lungs is worse than before, except for the limited consolidation. The end of the endotracheal tube is at the level of the T2 vertebral body

Later, we performed chest CT and identified that the end of the endotracheal tube was at the level of the lower edge of T1. Gradually, we found that the end of the endotracheal tube was at the level of the T1 vertebral body. Finally, we identified that the tip of the endotracheal tube was at the level of the T2 vertebral body. Hence, we clinically diagnosed the patient with severe pneumonia of the two lungs. Therefore, chest CT is recommended for further examination.

We identified mass-shaped high-density shadows on the right lung, and there were bronchial inflation shadows. Severe pneumonia in both of the lungs (Figure 2A). Compared with the previous film (2019-04-09), we found that the bipulmonary pneumonia had improved and better than before (Figure 2B). Compared with the previous film (2019-04-12), we identified that the bipulmonary pneumonia was heavier than before. The end of the endotracheal tube was at the level of the lower edge of T1 (Figure 2C). Compared with the previous film (2019-04-16), the bipulmonary pneumonia was heavier than before, and the tip of the endotracheal tube was at the level of the T1 vertebral body (Figure 2D). Compared with the previous film (2019-04-19), we found severe pneumonia of the two lungs was worse. Chest CT was recommended for further examination. The end of the endotracheal tube was at the level of the T2 vertebral body (Figure 2E).

Laboratory findings

We performed routine blood tests, protein and enzymatic analyses, immunopathological analysis, amino acid analysis, clotting factor analysis, hepatitis antigen-antibody analysis, viral and bacterial studies, bacterial and fungal cultures, bacterial DNA study, cellular immunoassay, G-experiment, endotoxin test, blood grouping, physical and chemical characteristics of blood and urine and stool culture of the patient. The results of each test were given in detail (Supplementary Table S1-S14).

Immunological findings

FACS results showed that the percentages of T cells (patient: 0.00%; controls: 60.8–75.4%) and B cells (patient: 0.00%; controls: 6.0–25.0%) present in the proband were significantly lower than those from the healthy family members. The percentage of CD3CD56+ NK cells were significantly higher (patient: 99.0%; controls: 5.0–20.0%). We further studied the subpopulation of T cells including CD4 and CD8. The percentage of CD4+ T cells (patient: 0.00%; controls: 29.4–45.8%) and CD8+ T cells (patient: 0.00%; controls: 18.2–32.8%) were significantly lower in the patient (Figure 3).

Figure 3 (A–P) FACS results showed that the percentages of T cells (patient: 0.00%; controls: 60.8-75.4%) and B cells (patient: 0.00%; controls: 6.0-25.0%) present in the patient were significantly lower than those from the family members. The percentage of CD3CD56+ NK cells were significantly higher (patient: 99.0%; controls: 5.0-20.0%). The subpopulation of T cells including CD4 and CD8. The percentage of CD4+ T cells (patient: 0.00%; controls: 29.4-45.8%) and CD8+ T cells (patient: 0.00%; controls: 18.2–32.8%) were also affected in the patient.

Immunoglobulin expression on B cells was also investigated. The levels of both IgA and IgM expression were considerably lower in the patient than in other healthy family members.

Whole-exome sequencing and Sanger sequencing identified a homozygous novel variant in RAG1

We performed WES of DNA from the proband. WES identified a novel homozygous single nucleotide deletion (c.2662delC), in exon 2 of the RAG1 gene in the proband (Figure 4). This novel homozygous variant leads to a frame-shift (p.Leu888Phefs*3) followed by premature translation termination, which finally results in the formation of a truncated RAG1 protein of 890 amino acids instead of the wild type RAG1 protein consisting of 1043 amino acids. Hence, it is a loss-of-function variant. Sanger sequencing confirmed that both the father and mother of the pro-band were harbouring this variant in a heterozygous state. Sanger sequencing also revealed that two elder sisters and one elder brother of the proband were phenotypically normal and lacked this variant. This variant is not found in 100 normal control individuals of the same ethnicity. This variant is also not present in the Human Gene Variant database (HGMD, www.hgmd.cf.ac.uk/), Online Mendelian Inheritance in Man (MIM, (https://www.omim.org). This homozygous novel variant is also not found in our in-house database, consisting of ~50,000 Chinese Han samples. We also did not find this variant in ExAC (exac.broadinstitute. org), gnomAD (https://gnomad.broadinstitute.org), dbSNP (https://www.ncbi.nlm.nih.gov/SNP) and 1000 Genome Database (www.internationalgenome.org).

Figure 4 Partial DNA sequences in the RAG1 gene by Sanger sequencing of the patient. The reference sequence NM_000448.2 of RAG1 gene was used.

In silico analysis

The variant (c.2662delC; p.L888Ffs*3) was predicted as “disease causing” by the Mutation Taster (http://mutation-taster.org/).19

Discussion

In 1965, OS was described and reported for the first time by Omenn, 1965.20 Although, pathogenesis of OS is complex, germline mutations in RAG1 gene is the genetic cause among most OS patients.16 In the present study, we analysed and investigated the clinical, immunological, and genetic characteristics of one patient with OS in our hospital. The affected child was carrying a frameshift mutation (c.2662delC, p.Leu888Phefs*3) in exon 2 of the RAG1 gene. This mutation was predicted to be deleterious and disease-causing. Furthermore, this novel frameshift mutation induced a premature stop codon and led to the formation of a truncated RAG1 protein.

RAG1 gene is significantly involved in the initiation of recombination process of the V, (D), and J segments, which finally form the variable portions of immunoglobulin and TCR proteins.9 Germline mutations in RAG1 gene causes profound reduction of T and B cells, leads to the occur-rence of OS.1 OS is extremely rarest types of SCID, usually manifested with gradually and progressively increased oligoclonal and activated T cells, with absence of B lymphocytes, which in turn results into the clinical phenotype including generalized erythroderma, lymphadenopathy, hepatosplenomegaly, eosinophilia, and elevated level of serum IgE. Unlike most of the OS cases with easy clinical diagnosis, some cases of maternal T cells engraftment within the foetus are really challenging. Hence, clinical diagnosis and treatment of OS patients is highly challenging without proper genetic screening. In Chinese population, the incidence rate of OS is very rare and only one OS patient has been reported so far.9 Here, we analysed and investigated the phenotype and genotype of a children with OS in a nonconsanguineous Chinese family.

To date, more than 50 variants of RAG1 gene have been reported in RAG1 mutation database (https://data-bases.lovd.nl/shared/variants/RAG1/unique?search_var_status=%3D%22Marked%22%7C%3D%22Public%22). Among these reported variants of the RAG1 gene, most are null or loss-of-function mutations.21 We reported a homozygous novel single nucleotide deletion in RAG1 gene, which led to a frameshift followed by the formation of a truncated RAG1 protein. Interestingly, the same RAG1 variants also cause different clinical manifestations.22 The human RAG1 is a highly conserved gene, homozygous or compound heterozygous mutation only causes OS in an auto-somal recessive mode of inheritance. According to the information provided by GnomAD, only 44% of amino acids in RAG1 is reported to cause OS upon mutation. However, functional characterization of the reported variants was very challenging.23 In addition, identifying variants based on low allele frequency is also not always correct since the RAG1 gene is highly conserved. OS patients with germline mutations in RAG1 gene usually presented with diverse phenotypes, and the treatment strategies are also highly variable among patients. Hematopoietic stem cell transplantation could be recommended for some OS patients, while mechanism-based treatment also became a good approach for other patient groups depending on their phenotypes.24,25

The patients with OS usually presented with decreased number of dysfunctional T cells, the complete absence of B cells, with normal counts of NK cells.26 Therefore, OS patients showed an immunological phenotype of TBNK+. Additionally, patients with OS also manifested significantly increased eosinophil counts and elevated serum IgE levels.27 Reduced or complete absence of both T and B cells leads to recurrent and severe infections in infancy for patients with OS. In patients with OS, loss of balance in the Th1/Th2 ratio causes increased secretion of IL-4 and IL-5 followed by elevated serum IgE levels, which finally results into eczema-like rashes.28,29 However, due to dysfunctional T-cells, patients with OS are usually identified with monoclonal TCR peaks. Dysfunctional T-cells and dysplasia of B-cells lead to cellular and humoral immune system abnormalities in patients with OS. Treatment with gamma globulin or antibiotic administration was not adequate or effective for patients with OS.

OS is a very rare and extremely heterogeneous (both genotypically and phenotypically) disorder. Single-gene sequencing or targeted next-generation sequencing does not always identify the candidate variants in patients with OS.30 Therefore, WES is more accurate and reliable for identifying the candidate gene and variants underlying the disease phenotype in patients with OS.31 WES is one of the most significant technologies for identifying candidate gene and disease-causing variants in patients with OS.32 WES is a more accurate, rapid and cost-effective tool for early and timely molecular genetic analysis allowing clinicians for making an accurate clinical diagnosis.33,34 Our present study not only expands the mutational spectrum of the OS gene but also strongly emphasizes the significance of WES as an accurate, rapid and cost-effective tool for molecular genetic analysis for patients with OS.

Acknowledgements

We are thankful to the proband and all the family members for participating in our study. We also thankful to the Gansu Health Scientific Research Project (GSWSKY2017-22) and Gansu Provincial Key Laboratory of Birth Defect Prevention and Control Research (1506RTSA158).

Conflict of Interest

The authors confirm that there are no conflicts of interest.

Author Contributions

Designed the study: Bin Yi, Ruifeng Xu, Zhaoyan Meng and Santasree Banerjee. Conducted acquisition and analysis of all the clinical data: Wendi Wang, Jian Wang, Jingjing Wang. WES pipeline and analysed the data: Jingting Liu, Jianying Pei, Wanyi Li. Selected patients and performed WES: Jianying Pei, Wanyi Li, and Yanxia Wang. Supervised manuscript preparation and edited the manuscript: Wendi Wang, Jian Wang, Jingjing Wang, Ruifeng Xu, Zhaoyan Meng, Bin Yi and Santasree Banerjee.

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Supplementary

Table S1 Routine blood test report of the patient.

Test Project 08/04/2019 12/04/2019 15/04/2019 18/04/2019 21/04/2019 Reference unit
leukocyte 4.36 2.42 ↓ 2.29 ↓ 2.26 ↓ 2.58 ↓ 4–11 109/L
Erythrocyte 4.6 3.8 3.6 ↓ 3.2 ↓ 3 ↓ 3.8–5.8 1012/L
Hemoglobin 116 ↓ 94 ↓ 89 ↓ 78 ↓ 71 ↓ 117–174 g/L
Haematocrit 37.1 29.8 ↓ 28.6 ↓ 25.1 ↓ 22.3 ↓ 35–51 %
Mean corpuscular volume 80.7 79 ↓ 79 ↓ 78.7 ↓ 75.1 ↓ 80–100 fL
Mean corpuscular haemoglobin 25.2 ↓ 24.9 ↓ 24.6 ↓ 24.5 ↓ 23.9 ↓ 27–35 pg
Mean erythrocyte haemoglobin concentration 313 315 311 311 318 310–370 g/L
Platelet count 769 ↑ 599 ↑ 711 ↑ 645 ↑ 631 ↑ 100–300 109/L
Lymphocyte percentage 27.1 12 ↓ 17.9 ↓ 8 ↓ 19.6 ↓ 20–40 %
Monocyte percentage 12.4 ↑ 3.7 5.7 18.1 ↑ 1.9 ↓ 3–8 %
Neutrophil percentage 58.9 76.9 ↑ 67.7 58.9 60.7 50–70 %
Eosinophil percentage 0.5 ↓ 6.6 ↑ 7 ↑ 2.7 7 ↑ 1–3 %
Basophil percentage 0.2 0 0.4 0.4 0 0–1 %
Lymphocyte absolute value 1.18 ↓ 0.29 ↓ 0.41 ↓ 0.18 ↓ 0.31 ↓ 1.5–4 109/L
Monocyte absolute value 0.54 ↑ 0.09 0.13 0.41 0.03 0–0.45 109/L
Neutrophil absolute value 2.57 1.86 ↓ 1.55 ↓ 1.33 ↓ 0.96 ↓ 2–7 109/L
Eosinophil absolute value 0.02 0.16 0.16 0.06 0.11 0–0.45 109/L
Absolute value of basophil 0.01 0 0.01 0.01 0 0–0.02 109/L
Red blood cell distribution width (SD) 48.1 ↑ 48.1 ↑ 48.9 ↑ 49.1 ↑ 48.8 ↑ 39–46 fL
Red blood cell distribution width (CV) 16.4 ↑ 16.6 ↑ 16.9 ↑ 17 ↑ 17.6 ↑ 11.6–14 %
Platelet distribution width 11.2 11.5 10.4 10.3 10.3 9.8–17.2 fL
Average platelet volume 10.2 10.7 9.7 9.5 9.7 6.5–12 fL
Large platelet ratio 26 28.5 22.3 21.1 22.7 13–43 %
Platelet specific product 0.79 0.64 0.69 0.61 0.61 %
Naive granulocyte absolute value 0.04 0.02 0.03 0.27 0.17 0–0.06
Naive granulocyte percentage 0.9 ↑ 0.8 ↑ 1.3 ↑ 11.9 ↑ 10.8 ↑ 0–0.6
Percentage of nucleated red blood cells 0 0 0 0 %
Absolute value of nucleated red blood cells 0 0 0 0 %
Serum amyloid 71.2 ↑ <5 87.52 ↑ 78.59 ↑ 0–10 mg/L

Table S2 Protein and Enzymatic Analysis of the patient.

Test Project 08/04/2019 12/04/2019 15/04/2019 18/04/2019 21/04/2019 Reference Unit
Alanine aminotransferase 33.5 22.8 29.6 34 34.9 0–40 U/L
Aspartate aminotransferase 70.8 ↓ 62.8 ↓ 110 ↓ 109.9 ↓ 113.1 ↓ 5–40 U/L
Asparagus 2.11 2.75 3.72 3.23 3.24 >1
Total protein 61.4 ↓ 49.3 ↓ 50.8 ↓ 54.1 ↓ 56.7 ↓ 66–87 g/L
albumin 38.6 30.3 ↓ 31.7 ↓ 27.3 ↓ 30.3 ↓ 35–55 g/L
globulin 22.8 19 ↓ 19.1 ↓ 26.8 26.4 20–40 g/L
White ball ratio 1.7 1.6 1.7 1 ↓ 1.1 1.1–2.5
Total bilirubin 5 ↓ 2.5 ↓ 2.6 ↓ 2.9 ↓ 1.9 ↓ 5.1–25 μmol/L
Direct bilirubin 2 0.8 1.2 0.4 0–10 μmol/L
Indirect bilirubin 3 1.8 ↓ 1.7 ↓ 1.5 ↓ 2–14 μmol/L
Alkaline phosphatase 130 ↑ 101 83 78 39–117 U/L
Lactate dehydrogenase 383 ↑ 433 ↑ 927 ↑ 619 ↑ 598 ↑ 114–245 U/L
γ-glutamyl transpeptidase 50 65 ↑ 78 ↑ 101 ↑ 11–50 U/L
Determination of adenosine deaminase (ADA) 18.9 26.2 19.8 19.9 U/L
Creatine kinase 37 ↓ 48 ↓ 261 35 ↓ 28 ↓ 150–325 U/L
Creatine kinase isoenzyme 19.8 17.4 75 ↑ 12.5 14.4 0–25 U/L
alpha-hydroxybutyrate dehydrogenase 320 ↑ 352 ↑ 774 ↑ 509 ↑ 484 ↑ 72–182 U/L
glucose 5.73 ↑ 4.95 4.23 5.87 ↑ 5.1 3.33–5.55 mmol/L
Urea nitrogen 2 1.8 1.6 1.2 2 mmol/L
Creatinine 21 ↓ 6 ↓ 11 ↓ 8 ↓ 7 ↓ 41–81 μmol/L
Uric acid 225 147 ↓ 164 ↓ 104 ↓ 179 ↓ 214–420 μmol/L
Determination of serum cystatin C 1.3 0.9 1.1 1 0.9 mg/L
Total cholesterol 1.86 ↓ 3.4–6.5 mmol/L
Triglyceride 1.4 0.34–1.92 mmol/L
High density lipoprotein cholesterol 0.46 ↓ 0.9–1.68 mmol/L
LDL cholesterol 0.71 ↓ 1.9–3.8 mmol/L
Apolipoprotein A1 0.6 ↓ 1.2–1.8 g/L
Glycolic acid 4.78 8.15 ↑ 14.02 ↑ 6 ↑ 0.4–5 mg/L
Retinol binding protein 14.4 ↓ 13.6 ↓ 21.3 ↓ 15.4 ↓ 16.9 ↓ 22–53 mg/L
Apolipoprotein B 0.5 0.5–1.5 g/L
calcium 2.21 ↑ 2.06 2.06 2.03 2.07 2.1–2.75 mmol/L
phosphorus 1.24 ↓ 1.5–2.2 mmol/L
iron 2.6 ↓ 7–19 μmol/L
Zinc 4.42 ↓ 7.72–21.4 μmol/L
magnesium 0.92 0.66–1.1 mmol/L
Potassium 4.71 4.73 5.75 ↑ 4.44 4.08 3.5–5.5 mmol/L
sodium 136 ↑ 132 ↓ 135 134 ↓ 136 135–145 mmol/L
chlorine 97 95 ↓ 91 ↓ 90 ↓ 92 ↓ 96–110 mmol/L
Amylase 16 0–200 U/L
Urea/creatinine 0.1 0.3 ↑ 0.15 0.15 0.29 ↑ 0.05–0.24
CO2 binding 22.9 23.1 35 ↑ 39.7 ↑ 33.8 ↑ 17.2–29 mmol/L
Immunoglobulin G 6.78 4.48 2.32–14.11 g/L
Immunoglobulin A 0 0 0–0.83 g/L
Immunoglobulin M 0.02 ↓ 0.03 ↓ 0.17–0.66 g/L
Procalcitonin (PCT) testing 1.2 ↑ 8.02 ↑ 1.44 ↑ 1.86 ↑ 0.94 ↑ 0–0.5 ng/ml
Superoxide dismutase 109.9 U/ml
Calcium ion 1.1 1.03 1.03 1.01 1.03 0.9–1.4 mmol/L
Complement C3 1.5 1.25 0.8–1.6 g/L
Complement C4 0.47 ↑ 0.45 ↑ 0.1–0.4 g/L
C-reactive protein 31.25 ↑ 16.55 ↑ 8.16 ↑ 22.36 ↑ 16.73 ↑ 0-5 mg/L
B-type brain natriuretic peptide precursor 100 100 150 0–300 pg/ml
Serum folic acid 26 3.89–26.8 ng/ml
Serum ferritin 537.3 ↑ 25–200 ng/ml
Vitamin B12 807.4 ↑ 100–700 pg/ml

Table S3 Immunopathological analysis of the patient.

Test Project 11/04/2019 15/04/2019 Reference Unit
Total T cells (CD3+) 0 ↓ 0 ↓ 60.8–75.4 %
T helper cells (CD3+ CD4+) 0 ↓ 0 ↓ 29.4–45.8 %
T suppressor cells (CD3+ CD8+) 0 ↓ 0 ↓ 18.2–32.8 %
Total B cells (CD3− CD19+) 0 ↓ 0 ↓ 6–25 %
NK cells (CD3-CD16+ CD56+) 98 ↑ 99 ↑ 5–20 %
T helper cells/T suppressor cells 0 ↓ 0 ↓ 0.9–3.6

Table S4 Amino acid analysis of the patient.

Test Project 12/04/2019 Reference
Alanine 150.63 ↓ 172–1053
Arginine 7.47 2–44
Citrulline 6.43 6–52
Glycine 212.3 190–1255
Leucine 108.02 79–350
Methionine 17.9 6–34
Ornithine 50.61 28–318
Phenylalanine 62.4 24–100
Proline 106.18 95–418
Tyrosine 45.71 40–278
Valine 106.45 63–298
ARG/ORN 0.15 0.02–0.64
CIT/ARG 0.86 0.09–9.86
MET/PHE 0.29 0.07–4.11
ORN/CIT 7.87 0.95–16.13
PHE/TYR 1.37 ↑ 0.18–1.09
TYR/CIT 7.11 0.02–21.9
ORN/PHE 0.81 0.15–4.83
LEU/PHE 1.73 0.49–6.88
GLY/PHE 3.4 4.19–20.39
Free carnitine 31 10.5–67.8
Acetyl Carnitine 31.56 1.3–39.3
Propionylcarnitine 0.96 0.3–4.95
C3DC-C4OH 0.21 0.02–0.36
Butyrylcarnitine 0.24 0.08–0.51
C4DC-C5OH 0.27 0.09–0.55
Isovalerylcarnitine 0.11 0.05–0.53
Prenylcarnitine 0.01 0.00–0.03
C5DC-C6OH 0.1 0.05–0.41
Hexanoylcarnitine 0.14 ↑ 0.01–0.13
Adipylcarnitine 0.09 0.03–0.29
Octanoylcarnitine 0.09 0.01–0.14
Octenoylcarnitine 0.2 0.01–0.57
Decanoylcarnitine 0.11 0.02–0.19
Decenoylcarnitine 0.1 0.02–0.21
Decadienoylcarnitine 0.02 0.01–0.21
Dodecylcarnitine 0.07 0.02–0.22
Dodecenoylcarnitine 0.06 0.01–0.26
Myristyl carnitine 0.12 0.05–0.39
Myristyl carnitine 0.08 0.02–0.29
3-hydroxy-tetradecanoylcarnitine 0.01 0–0.03
Tetradecadienoylcarnitine 0.02 0.01–0.07
Cetyl carnitine 0.78 0.47–6.34
3-hydroxy-hexadecanoylcarnitine 0.03 0.01–0.05
Hexadecenoylcarnitine 0.09 0.02-0.39
3-hydroxy-hexadecenoylcarnitine 0.03 0.01-0.13
18 carbonyl carnitine 0.41 0.21-1.68
3-hydroxy-octadecanoylcarnitine 0.01 0-0.03
18 carbenoyl carnitine 1.38 0.39-2.75
3-hydroxy-octadecenoyl carnitine 0.02 0.01-0.06
18 carbon dienoyl carnitine 0.27 0.07-0.66
C3/C2 0.03 ↓ 0.05-0.27
C4/C2 0.01 0-0.1
C5/C2 0 0-0.11
C8/C2 0 0-0.03
C14: 1/C16 0.1 0.01-0.21
C16-OH/C16 0.04 ↑ 0-0.03
C14: 1/C8: 1 0.06 0.1-2.27
CO/ (C16+ C18) 26.05 2.56-48.97
C3/Met 0.07 ↓ 0.12-1.99
C3/CO 0.03 0.01-0.15
C4/C3 0.25 0.04-0.38
C5/CO 0 0-0.02
C5/C3 0.11 0.03-0.51
(C3DC-C4OH) /C4 0.88 0.14-1.59
(C4DC-C5OH) /CO 0.01 0-0.02
(C4DC-C5OH) /C8 3 1.39-18.08
(C5DC-C6OH) /C8 1.11 0.83-14
C14: 1/C12: 1 0.15 ↑ 0.01-0.12
C10/C8 0.82 0.38-2
C5DC-C6OH/C3 0 0-0.06
C16/C2 0.02 ↓ 0.06-1.33
C16/C3 0.81 0.4-5.61
C18/C3 0.43 0.14-1.69
C18OH/C3 0.01 0-0.03

Table S5 Clotting factor analysis of the patient.

Test Project 09/04/2019 12/04/2019 References Unit
Prothrombin time 34.7 ↑ 11.6 0–14 s
Prothrombin time activity 21.9 ↓ 108.6 70–130 %
Prothrombin time ratio 3.04 ↑ 0.97 0.85–1.15
International normalized ratio of prothrombin time 3.04 ↑ 0.97 0.8–1.5
Activated partial prothrombin time 80 ↑ 52.8 ↑ 22–40 s
Fibrinogen assay 2.78 2.46 2–6 g/L
Thrombin time determination >60 ↑ 24.1 11–25 s

Table S6 Hepatitis antigen-antibody analysis for the patient.

Test Project 09/04/2019
Hepatitis B surface antigen Negative (−)
Hepatitis B surface antibody Positive (+)
Hepatitis B e antigen Negative (−)
Hepatitis B e antibody Negative (−)
Hepatitis B core antibody Negative (−)
Pre-si antigen of hepatitis B virus outer membrane protein Negative (−)
Hepatitis C antibody Negative (−)
Hepatitis E antibody Negative (−)
AIDS antibody Negative (−)
Treponema pallidum antibody Negative (−)

Table S7 Virus and bacterial study of the patient.

Test Project 09/04/2019
Parainfluenza virus IgM antibody Negative (−)
Syncytial virus IgM antibody Negative (−)
Mycoplasma pneumoniae IgM antibody Negative (−)
Chlamydia pneumoniae IgM antibody Negative (−)
Adenovirus antibody assay Negative (−)
Influenza virus antibody assay Negative (−)

Table S8 Bacterial and fungal culture analysis of the patient.

Test Project 09/04/2019 10/04/2019 16/04/2019
Bacterial culture and identification See normal respiratory bacteria No bacterial growth See normal respiratory bacteria
Fungal culture identification Candida albicans No fungal growth No fungal growth

Table S9 Bacterial DNA study in the patient.

Test Project 11/04/2019 15/04/2019 Reference
Mycoplasma pneumoniae-DNA Negative (−) Negative (−) Negative
Chlamydia pneumoniae-DNA Negative (−) Negative (−) Negative
Mycobacterium tuberculosis-DNA Negative (−) Negative (−) Negative
Streptococcus pneumoniae DNA Negative (−) Negative (−) Negative
Staphylococcus aureus DNA Negative (−) Negative (−) Negative
Escherichia coli DNA Negative (−) Negative (−) Negative
Klebsiella pneumoniae DNA Negative (−) Negative (−) Negative
Pseudomonas aeruginosa DNA Negative (−) Negative (−) Negative
Acinetobacter baumannii DNA Negative (−) Negative (−) Negative
Stenotrophomonas maltophilia DNA Negative (−) Negative (−) Negative
Haemophilus influenzae DNA Negative (−) Negative (−) Negative
Legionella pneumophila DNA Negative (−) Negative (−) Negative
Methicillin-resistant Staphylococcus DNA Positive (+) Positive (+) Negative
Mycobacterium tuberculosis complex DNA Negative (−) Negative (−) Negative

Table S10 Cellular immunoassay of the patient.

Test Project 12/04/2019
No stimulation (N) 0.03
Non-specific gamma interferon (P) 0.02
Specific gamma interferon (T) 0.02
(T-N) / (P-N) 0.2
Mycobacterium tuberculosis specific cellular immunoassay Negative

Table S11 G-experiment and endotoxin test for the patient.

Test Project 15/04/2019 Reference Unit
G experiment 58.47 <70 pg/ml
GM experiment 0.52 <0.65 ug/ml
Endotoxin test 0.06 <0.08 EU/ml

Table S12 Blood grouping of the patient.

Test Project 22/04/2019 22/04/2019
ABO blood type O type O type
RH blood type Positive Positive
ABO blood type (negative) O type
Irregular antibody screening negative

Table S13 Physical and chemical characteristics of patient’s blood and urine.

Test Project 13/04/2019 Reference Unit
pH 6.5 5.4–8.4
proportion 1.005 1–1.03
protein negative negative g/L
glucose negative negative mmol/L
Ketone body negative negative mg/L
Bilirubin negative μmol/L
Urobilinogen normal μmol/L
Nitrite negative negative
Urine leukocyte negative negative leu/μl
Occult blood negative negative mg/l
ascorbic acid negative mmol/L
colour Light yellow
transparency Clear
leukocyte 13 ↑ 0–11 μL
Leukocyte mass 0–2 /μl
Atypical red blood cells %
Erythrocyte 1 0–9 μL
Squamous epithelium 0–11 /μl
Renal epithelium 0–6 /μl
Oval fat body 0–2 /μl
Mucus 0–480 /μl
Transparent tube type 0–2 /LPF
Granular cast 0–1 /LPF
Erythrocyte cast 0–1 /LPF
Leukocyte cast 0–1 /LPF
Epithelial cell cast 0–1 /LPF
Cell cast 0–1 /LPF
Wide tube type 0–1 /LPF
Fat tube type 0–1 /LPF
Wax type 0–1 /LPF
Unclassified cast 0–2 /LPF
Calcium oxalate crystal 0–3 /HPF
Calcium phosphate crystal 0–3 /HPF
Calcium carbonate crystals 0–3 /HPF
Urate Crystal 0–3 /HPF
Unclassified crystal 0–10 /HPF

Table S14 Stool culture of the patient.

Test Project 14/04/2019 Reference Unit
Stool color yellow
Fecal traits Soft stool
leukocyte Not seen 0–3 Unit/HP
Erythrocyte Not seen 0 Unit /HP
Fecal occult blood Negative (−) negative
Fat ball Not seen Not seen Unit /HP
phagocyte Not seen Not seen Unit /HP
Pus cell Not seen Not seen Unit /HP
Starch granules Not seen Not seen Unit /HP
Yeast-like bacteria Not seen Not seen Unit /HP
Intestinal mucosal epithelial cells Negative (−) Not seen Unit /HP
Plant cell Not seen Not seen Unit /HP
Fungus Not seen Not seen Unit /HP
Pinworm eggs Not seen Not seen Unit /HP
Roundworm eggs Not seen Not seen Unit /HP
Whipworm eggs Not seen Not seen Unit /HP
Taenia solium Not seen Not seen Unit /HP
protozoan Not seen Not seen Unit /HP
Muscle fibre Not seen Not seen Unit /HP
Connective tissue Not seen Not seen Unit /HP