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An immunocompetent patient with a nonsense mutation in NHEJ1 gene



DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage. DSBs are repaired by homologous recombination or non-homologous end-joining (NHEJ). NHEJ, which is central to the process of V(D)J recombination is the principle pathway for DSB repair in higher eukaryotes. Mutations in NHEJ1 gene have been associated with severe combined immunodeficiency.

Case presentation

The patient was a 3.5-year-old girl, a product of consanguineous first-degree cousin marriage, who was homozygous for a nonsense mutation in NHEJ1 gene. She had initially presented with failure to thrive, proportional microcephaly as well as autoimmune hemolytic anemia (AIHA), which responded well to treatment with prednisolone. However, the patient was immunocompetent despite having this pathogenic mutation.


Herein, we report on a patient who was clinically immunocompetent despite having a pathogenic mutation in NHEJ1 gene. Our findings provided evidence for the importance of other end-joining auxiliary pathways that would function in maintaining genetic stability. Clinicians should therefore be aware that pathogenic mutations in NHEJ pathway are not necessarily associated with clinical immunodeficiency.

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DNA double-strand breaks (DSBs), resulting in loss of considerable chromosomal regions, are among the most deleterious types of DNA damage. With an estimated rate of ten per day, DSBs can either be caused by DNA damaging agents such as reactive oxygen species or could be a part of physiological DNA recombination taking place in the immune system [1, 2]. V(D)J recombination is a process during which the highly diverse lymphocyte antigen receptors breed. One of the consequences of this process is DSB [3]. If left unrepaired, DSB will induce either apoptosis or cellular dysfunction [4].

DSBs are repaired by homologous recombination or non-homologous end-joining (NHEJ) [5]. Error-prone NHEJ, which is central to the process of V(D)J recombination, is the main pathway of DSB repair system in higher eukaryotes [5, 6]. Considering its major role in the immune system development, deficiency of NHEJ1 gene products manifests with absence of mature T and B lymphocytes, also known as “severe combined immunodeficiency” (SCID) [7]. Mutations in the NHEJ1 have been associated with the clinical phenotype of severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation (Phenotype MIM # 611291). The patients reported so far have always presented with clinical manifestations such as failure to thrive (FTT), severe growth retardation, microcephaly, and autoimmune hemolytic anemia (AIHA) [8,9,10]. Herein, we report on a patient who was clinically immunocompetent despite having a pathogenic mutation in NHEJ1 gene.

Case presentation

The patient was a 3.5-year-old girl, a product of consanguineous first-degree cousin marriage, who was born at the gestational age of 38 weeks after a normal and uncomplicated pregnancy. She was in good health after delivery with a good APGAR score. Her weight, length and head circumference were 2500 g, 45 cm and 33 cm, respectively. Weight and length were below the 3rd percentile, whereas head circumference was slightly above the 15th percentile according to the national child growth curve. Failure to thrive and proportional microcephaly continued until one year of age but development was good. She presented with jaundice at the age of one year. Laboratory tests showed decreased WBC count (3000/mm3, reference range for age: 5000–15,500/mm3) with 64% neutrophil and decreased hemoglobin levels (11 g/dL, reference range for age: 12–14 g/dL). Furthermore, lab results revealed an MCV of 88.7 fL, platelet count of 261,000, ESR of 2 mm/h, reticulocyte count of 5.1%, a positive direct Coomb’s test, negative indirect Coomb’s test. Moreover, ACLA, ANA, ds-DNA, C3, C4, ANCA were within normal range. Osmotic fragility test was negative. Hb electrophoresis showed Hb-A1 of 91.8%, Hb-F of 5.7%, and Hb-A2 of 2.5%. Viral marker tests revealed negative cytomegalovirus (CMV) PCR and parvovirus antibody. The patient was referred to a hemato-oncologist with a diagnosis of AIHA and was subsequently treated with prednisolone. The patient’s parents did not mention any history of hospitalization or outpatient visits due to infectious disorders. Furthermore, according to her flow-cytometry results, low level of CD19+ and the very high level of CD56+ cells were detected. (Table 1). The immunophenotyping test were performed at the age of 2.5 years.

Table 1 The results of flow-cytometry and blood count

As the patient was a result of a consanguineous marriage, a thorough family history was taken from her parents. Both her parents were in good health. The other sibling was a boy who presented with jaundice and anemia at the age of three months. He then presented with recurrent infections and passed away at the age of three years due to pneumonia. Serum PCR for CMV was positive in the deceased individual. No further clinical and laboratory data were available.

To evaluate the patient for the underlying genetic disorder, whole-exome sequencing was carried out on the DNA extracted from the proband’s peripheral blood sample. Whole Exome Sequencing (WES) was performed on Illumina NextSeq500 instrument. The sequencing results were subsequently analyzed using different bioinformatics tools and databases such as BWA aligner, GATK and ANNOVAR. Whole exome sequencing details of coverage and number of reads are provided in Table 2. It was found that the patient had a stop-gain mutation in NHEJ1 gene (NM_024782.2:c.532C > A). Sanger sequencing subsequently confirmed that the patient was homozygous and both parents were heterozygous for the mutation (Fig. 1).

Table 2 Whole Exome Sequencing detail of coverage and number of reads
Fig. 1

WES results showing homozygous mutation in NHEJ1 gene in the proband, as visualized using Integrative Genome Viewer (IGV). Sanger sequencing confirms the presence of heterozygous mutation in NHEJ1 gene in both parents. Arrows indicate the site of the mutation

On follow-up, the patient had growth and development retardation with her length/height, head circumference and weight being below the 3rd percentile corrected for the age. Except for axillary lymphadenitis following BCG vaccination, the patient had had full vaccination including BCG, HepB, polio, MMR and DTP without any complications. On the last follow-up at the age of three years, the patient’s height and weight were 86 cm and 9.1 kg, respectively—both below the 3rd percentile corrected for the age. However, she has not had any evidence of immunodeficiency, despite living a normal life without any special precautions to preserve the patient’s health.

Discussion and conclusion

Human cells are exposed to a wide variety of endogenous and exogenous DNA damaging agents. DSBs are considered one of the most severe forms of DNA damage, which could lead to cellular apoptosis or carcinogenesis, if left unrepaired [4, 6, 8]. DSBs are mostly repaired by either homologous recombination or non-homologous end-joining pathway [5]. Animal models evaluating the role of NHEJ pathway in the immune system have highlighted its importance in the immune system [11]. Animal models with defects in NHEJ have B and T lymphocyte maturation arrest and even embryonic lethality when accompanied by deficiencies in XRCC4/DNA-Ligase IV complex [12,13,14]. Given the vital role of repairing DSBs, mutation in any of the NHEJ genes, cause disruption in the immune system development, particularly B cell and T cell maturation, resulting in SCID [6, 8]. SCID presents early during the first few months of life and displays with severe bacterial and opportunistic infections, particularly respiratory infections [9].

In addition, it has been proposed that NHEJ1 has an important role in human cerebral cortex development [15]. Decreased expression of NHEJ1 has been shown to lead to defects in neuronal migration and decreased width of external cortical layers [4]. Furthermore, NHEJ deficiency appears to be a risk factor for the development of malignancy. Defects in NHEJ in P53-deficient mice have been shown to perpetually lead to development of pro-B cell lymphomas [16].

During the embryonic period, all cells are in a hyper-mitotic state. Therefore, mutations in NHEJ1 commonly affect different cell types. Microcephaly, severe growth retardation, dysmorphic facial features and autoimmunity are reported alongside immunodeficiency [8, 17, 18]. To the best of our knowledge, the patient reported here is the first case with a homozygous pathogenic nonsense mutation (CADD score: 37) in NHEJ1 gene with a competent immune system. This mutation would lead to the production of a protein lacking about one-third of its C-terminal amino-acid sequence.

We reported a patient with a pathogenic stop-gain mutation in NHEJ1 who presented with AIHA, failure to thrive and microcephaly. However, she had no history of any bacterial or opportunistic infections. No previous history of respiratory infections, chronic diarrhea or any other complains was mentioned by her parents. The patient reported here had no history of prior hospital admission other than the one mentioned due to severe anemia. Buck, et al, reported a patient of Turkish origin with a similar mutation presenting with microcephaly and growth retardation and recurrent bacterial and opportunistic infections who had died at the age of four years due to septic shock [6]. Our findings highlight the importance of other end-joining auxiliary pathways such as polymerase θ-mediated end-joining, also known as a-EJ pathway [19, 20]. Although, due to the scarce number of individuals with deficiency in NHEJ, the exact role and function of a-EJ pathway is largely unknown, recent studies have emphasized its role in sustaining cell viability and genetic stability in case NHEJ is compromised.

All patients with defects in NHEJ reported to date, have been immunocompromised. Flow-cytometry in these patients demonstrates low T cell, very low or absent B cell and normal NK cell count. Serum immunoglobulin levels in these patients are generally low for IgG and IgA, and normal or high for IgM [3, 9, 18]. Our Patient had leukopenia with low level of CD19+ cells and very high level of CD56+ cells. In addition, IgA levels were mildly elevated. These findings might be a novel presentation. Further investigations to shed light on how these findings are compatible with immunocompetent phenotype are warranted. FTT and severe growth retardation have reported in all cases of NHEJ mutation [10]. Our patient’s weight, length/height and head circumference were below the 3rd percentile at birth and has been below the 3rd percentile on follow-up. Growth chart demonstrated that her growth pattern was not steady, as there were multiple periods of growth arrest. Her development was otherwise normal.

Microcephaly has been widely reported in previous studies, indicating the role of NHEJ gene in cerebral expansion [8]. NHEJ gene mutation leads to apoptotic death of post-mitotic neurons, causing CNS development issues, presenting itself with microcephaly, psychomotor retardation and ataxia [7, 17]. Our patient’s head circumference has always been below the 3rd percentile corrected for age. Her neurological development was otherwise normal and no finding in favor of developmental delay was noticed in her history or physical examination.

Live vaccines are absolutely contraindicated in patients with SCID as life-threatening complications could occur [21]. Our patient had complete vaccination record according to the national protocol including BCG, OPV and MMR without any complications except mild axillary lymphadenitis after BCG vaccination, which had resolved without medical intervention. Notably, vaccination against smallpox, chickenpox, influenza, and rotavirus is not part of Iran vaccination program.

Autoimmunity is common in previously reported patients with mutations in NHEJ genes; autoimmune cytopenia in particular is reported to be present in about a quarter of the patients [3, 22, 23]. The patient presented here was initially diagnosed with AIHA, but responded very well to oral prednisolone and required no further interventions.

In conclusion, our report highlights the importance of pathways other than NHEJ in DSB repair. Therefore, clinicians should be aware that pathogenic mutations in NHEJ pathway are not necessarily associated with clinical immunodeficiency.



Anticardiolipin antibody


Autoimmune hemolytic anemia


Antinuclear antibody


Anti-neutrophil cytoplasmic antibody


Bacillus Calmette–Guérin




Double-strand break


Double stranded DNA


Diphtheria, tetanus and pertussis


Failure to thrive


Measles, mumps, and rubella


Non-homologous end-joining


Severe combined immunodeficiency


White blood cell


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The authors would like to thank the family members for participating in this study.


This study was partly supported by the US NIH NINDS R01NS081208-01A1 awarded to MAF. The study was also partly supported by the NIMAD research grant (940714) awarded to MAF. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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All data are available from the corresponding author on request.

Author information




MAF conceived and designed the study, collected, assembled, interpreted NGS data. HE, MRB and ZH clinically evaluated the patient. HE and PH drafted the manuscript. HE, PH and MAF revised the manuscript. DA, MM and MF did the genetic studies. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mohammad Ali Faghihi.

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Ethics approval and consent to participate

The Ethics Committee of the Persian BayanGene Research and Training Center approved the study protocol. The parents signed a written informed consent to participate in this study. Written informed consent was obtained from the parents of the patient for the publication of this case report.

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The parents of the affected patient have consented to the publication of the case and accompanying clinical and genetics data.

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The authors declare that they have no competing interests.

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Esmaeilzadeh, H., Bordbar, M.R., Hojaji, Z. et al. An immunocompetent patient with a nonsense mutation in NHEJ1 gene. BMC Med Genet 20, 45 (2019).

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  • Severe combined immunodeficiency
  • Nonhomologous end-joining factor 1, human
  • Autoimmune hemolytic Anemia
  • Genetic disorders
  • Immunologic deficiency syndromes