A four year old with immunodeficiency

Patient Presentation

A severely undernourished 4-year-old girl is referred to the paediatric outpatient clinic for recurrent anaemia, an erythematous rash on her face and trunk and accompanying hepatosplenomegally.

Acknowledgment

This case study was kindly provided by Dr R Thejpal, Dr Y Goga, Dr B Neethling and Dr F Mamdoo, Specialists at Inkosi Albert Luthuli Hospital and also affiliated to the Department of Paediatrics, UKZN.

History

The child was born term, normal vaginal delivery. Although no complications were noted, her Apgar scores were recorded as 5, 6 and 8 at birth, 5 and 10 minutes respectively. Since birth the child was noted to have dysmorphic features that include most notably hypertelorism and small palpebral fissures. Her mother denies any history of antenatal alcohol use or any infections during her pregnancy. This was the first pregnancy and child born to both parents. The child was breastfed until 4 months of age; thereafter she was put on formula feeds and started on solid food by 9 months.

Her developmental milestones were delayed for both gross and fine motor skills and verbal development.

Past medical history

The child had been admitted to hospital at age 2½ years for lower respiratory tract infection (LRTI), suspected of being TB. The child was also failing to thrive, falling below the 5th centile for both height and weight. At this time she was investigated for TB, Epstein- Barr Virus (EBV), Toxoplasmosis, Rubella, Cytomegalovirus (CMV), Herpes, Hepatitis B and HIV. She was found to be negative for all investigations except EBV, which was IgM negative but IgG positive. She was further tested for Ceruloplasmin 0.69 (0.18-0.45) and copper levels 35.9mmol/l, which were both mildly elevated. Her complement C3 was 2.2 (0.8-1.5) and C4 was 0.90 (0.84), which were both elevated.

A year later at age 3½ years she was re-admitted for LRTI, gastric washings were submitted but found to be TB negative, while complement C3 and C4 levels remained elevated at the same levels recorded a year earlier.

Past surgical history

Nil noted

Family history

No positive TB contacts. Both parents are HIV negative and in good health.

No siblings or other children in the home

Travel history

None

Social history

Father is employed as a security guard and mother is currently unemployed. They live together in a one bedroom apartment with electricity and running water. No pets and no smoking in the home

Medication

Nil

Allergies

Nil known

Differential Diagnosis

• HIV
• TB
• Recurrent pneumonia
• Cystic fibrosis
• Hypogammaglobulinaemia
• Agammaglobulinaemia
• Severe combined immunodeficiency (SCID)

Examination

General

• Weight 8.3 kg, below 5^th centile
• Height 75cm, below 5^th centile
• Afebrile
• Blood pressure and heart rate normal

• Moderate pallor
• Generalised lymphadenopathy with some firm axillary lymph nodes bilaterally
• No dehydration
• No jaundice
• No stigmata of HIV

Head and Neck

• Dysmorphic features- hypertelorism and small palpebral fissures
• Puffy eyelids
• Anterior and posterior fontanelles open
• Hair has red discolouration and straight
• Erythematous rash with mild induration noted on parts of the face, forehead and trunk- thought to be atypical erythema nodosum

Abdomen

• Distended, with some scratch marks noted
• Soft and non tender on palpation
• Large hepatosplenomegaly
• Bowel sounds heard on auscultation

Respiratory

• No signs of respiratory distress
• Chest clear

Cardiovascular

• Ejection systolic murmur but no significant cardiac lesions

Neurological

• Delayed milestones with regards to motor skills (fine and gross) and verbal skills. Higher function could not be assessed because child did not communicate adequately
• Reflexes 2/4
• Sensation intact
• Tone normal
• Strength normal
• Gait normal

Musculoskeletal

• Areas of induration under the skin of both calves, possibly due to calcification in the calf muscles.

Investigations

Interpretation: T and B cell lymphopaenia noted

Preservation of natural killer cells

Suggestive of a primary immune deficiency

Current Admission

Bone marrow:

The aspirate failed, but the trephine was suggestive of mycobacterial infection with secondary marrow fibrosis. In addition, one of the TB cultures were Zn positive, however the final culture yielded no growth (could be MOTTS or MTB).

Liver biopsy:

Mild portal tract inflammation with eosinophils, extramedullary haemopoeisis and focal lobular inflammation; Mild macrovesicular steatosis; Liver biopsy result is in keeping with mild portal tract chronic inflammation with one non-necrotising granuloma on deeper sections, Ziehl Nielsen stains for AFB negative.

Skin Biopsy:

Skin with deep seated histiocytic infiltrate within the subcutaneous tissue. CD1a and S100 are negative excluding a Langerhans cell infiltrate; Chloroacetate esterase stain is positive in some of the immature myeloid cells. Stains for AFB and fungi are negative.

Chest X-ray:

Pneumonitis of both lung fields

Discussion

This case explores the clinical manifestation of primary immunodeficiency, specifically examining a type of severe combined immunodeficiency (SCID). SCID is a life-threatening syndrome of recurrent infections, diarrhoea, skin infections and failure to thrive that results from numerous molecular defects leading to severe T- and B-cell dysfunction and occasionally can affect natural killer (NK) cells. Without intervention, the T and B-cell dysfunction usually results in severe infection and death in children by age 2 years.

Suspected SCID is defined as less than 0.3×10⁹/L CD3 T cells or less than 20% of CD3/CD4 cells with naïve cell markers with one or more of the following: (1) abnormal TRECs (T-cell receptor excision circle) upon new born screening or presentation, (2) family history of SCID and (3) recurrent or opportunistic infections. Features of Omenn syndrome is also an early indicator (Severe Combined Immunodeficiency (SCID) | NIH: National Institute of Allergy and Infectious Diseases, 2015.). Clinically, most patients present before age 3 months with unusually severe and frequent infections by common or opportunistic pathogens.  This is obviously not the situation with our patient, but we will explore the mechanism and pathophysiology of SCID so that a likely scenario can be proposed with the 4-year-old girl in our case. It is first important to understand T and B-cells and the way in which they generate immunological diversity in a normally functioning immune system. All mechanisms described here are carefully illustrated in stepwise graphics to assist with understanding the processes described.

Figure 1. T and B cell V(D)J recombination. The image illustrates the specific tissue in which B and T cell recombination occurs, allowing BCR and TCR to recognize a wide range of pathogenic epitopes. Variability in epitope binding potential of the receptors is achieved by varying the combination of pre-existing multicopy gene segments called variable (V), diversity (D) and joining (J) segments found in the BCR-heavy and TCR-β and -δ chains. In B cells the process take place in the bone marrow and for T cells in the thymus. Created with biorender.com (CA Petersen 2024)

How is diversity normally generated in T and B cells?

T and B cells, but not NK cells, undergo V(D)J recombination in order to generate diverse repertoires of T and B cell receptors (TCR and BCR) capable of recognising a wide range of pathogens. This is achieved by each TCR and BCR being highly specific for small protein fragments derived from the pathogen. These small protein fragments are known as epitopes. As there are potentially an infinite number of epitopes, the TCR and BCR need to be adaptable and variable so that enough diversity is created to engage with each different epitope.

Figure 2. Illustrates how VDJ recombination occurs in the BCR-H, TCR-β and -δ chains. The BCR-L, TCR-α and -γ chains lack D segments and only undergo VJ recombination. Recombination activating genes (RAG-1 and RAG-2) mediates the recognition and splicing of the V, D and J segments. Created with biorender.com (CA Petersen 2024).

Variability in the epitope binding potential of the receptors is achieved by varying the combination of pre-existing genes known as Variable (V), Diversity (D) and Joining (J) segments. BCR (B cells) variability takes place in the bone marrow and TCR (T cells) variability occurs in the thymus. Variability and generation of diversity occurs by V(D)J gene recombination in the different anatomical locations. Expression of these recombined genes in the B cell makes up the protein structure of the BCRheavy chain and in the T cell makes up the TCR-b and -d chains. Diversity (D) segments are absent in the BCR-light (k and l) and in the TCR-a and -g chains, so only V and J recombination occurs for these proteins.

Figure 3. The RAG1/2 complex mediates the random selection and alignment of a D and J or V and DJ gene segment and introducing a double strand break in the DNA. The blunt-ends of the excision circle are ligated between the 12-RSS and 23-RSS sequences to generate the signal joint, while the 5’ and 3’ ends of the genomic DNA strands are covalently linked to generate hairpin stem loop structures. Created with biorender.com (CA Petersen 2024).

Let’s look a little closer at V(D)J recombination

VDJ recombination occurs in the BCR-H, the TCR-β and -δ chains, whilst only VJ recombination occurs in the BCR-L (k and l) and TCR-a and -g chains. Gene recombination can occur because of Recombination Activating Genes that encode two enzymes: RAG1 and RAG2.  These mediate the binding and splicing of V, D and J segments. RAG1 binds to specific recombination signal sequences (RSS) present as 23-RSS or 12RSS sequences, which flank each gene segment, and then recruit RAG2. Recombination can only occur between a 23-RSS and 12-RSS sequence (which is called the 12/23 rule). A double-stranded break is introduced into DNA followed by recruitment of DNA repair enzymes that mediate Non-Homologous End-Joining (NHEJ). Recognition and end-binding is mediated by proteins Ku70, Ku80 which recruit DNA-PKcs.

Figure 4. SCID: defective T and B cell development, normal NK cells. In certain types of SCID, defect in the development of T and B cells, but normal NK cells function is evident. This stems from defects in the V(D)J recombination processes common to T and b cell maturation, but is not required for NK cell development. Created with biorender.com (CA Petersen 2024).

There are many proteins involved in V(D)J recombination and to date genetic mutations in RAG1, RAG2, DNA-PKcs, DCLRE1C (also known as Artemis), XLF (also known as Cernunnos) and DNA ligase IV have been identified in the SCID phenotype that result in a lack of T and B cells, but with normal NK cell activity. The most common mutation is seen in RAG1 and RAG2.

According to the Primary Immune Deficiency Treatment consortium (PIDTC) 2022 definition for the diagnosis of SCID, there are three major subtypes: typical SCID with very low T cells, leaky SCID with low T cells and Omenn syndrome. Typical SCID describes patients with the most profound defect in host T cells, usually due to null pathogenic variants in genes whose products are essential for T cell development. Defects in 7 known genes (IL2RG, RAG1, RAG2, ADA, DCLRE1C IL7R and JAK3) represents 80% of SCID cases. A pathogenic finding in many genotypes of typical SCID is the presence of maternal T cells in the peripheral blood, this is due to the failure to reject transplacental transferred cells. Transplacental acquired maternal engraftment (TME) is approximately observed in 50% of patients with typical SCID but somewhat less in patients with a genetic subtype ADA, RAG1, RAG2 and DCLRE1C, which is likely due to the ability of NK cells and residual host T cells to eliminate the maternal cells. TME may elevate the total amount of T cells, so in the absence of TME the threshold for typical SCID diagnosis would less than 0.05 x10⁹/L CD3 T cells according to the PIDTC 2022 (Dvorak et al., 2023).

Figure 5. V(D)J recombination: RAG1 and RAG2 binding. V(DJ recombination begins with the binding of recombination activating gene 1 (RAG1) proteins to specific recombination signals sequences (RSS) present in each V, D and J segment. RAG2 is then recruited and the RAG1/2 complex mediates the formation of a double stranded beak in DNA which will allow a precise fusion if a D and J or V and (D)J segment. Created with biorender.com (CA Petersen 2024)

Worth noting is that in certain rare forms of SCID, with defects in T and B cell development, the genetic mutations in the affected proteins may permit partial enzymatic activity, which is then known as “leaky” SCID. Defects in RAG1, RAG2, ADA and RMRP are overrepresented in leaky SCID. Leaky SCID diagnosis requires at least 2 of the following: low T cell number s for age (<0.6×10⁹/L for any age, <0.8×10⁹/L if aged 2-4 years or <1.0×10⁹/L if aged < 2 years), an oligoclonal T cell population and low percentage of naive T cells or low/undetectable TRECs. It is also critical to test for TME, because maternal cells would classify the patient as typical SCID (Dvorak et al., 2023)

Figure 6. No- homologous end joining: recognition and end binding. Following the introduction of double stranded break in the genomic DNA RAG1/2 complex, recruitment of DNA repair enzymes is initiated to mediate non-homologous end joining (NHEJ) of DNA ends. Recognition and end-binding is first mediated by proteins Ku70, Ku80 which recruit DNA-PKs. *In SCID phenotypes with defective T and B cell development, but normal NK cell function, genetic mutation in DNA-PKcs have been identified Created with biorender.com (CA Petersen 2024)

It has been reported in Omenn’s syndrome that clonal expansion of activated T and B cells occurs and that blood levels of these cells may appear within normal ranges, or be elevated. Closer examination the TCR and BCR in these patients reveal them to be oligoclonal in nature. Oligoclonal refers to there only being very few BCR or TCR types because there is an inability to undergo complete V(D)J recombination, and the result is translated to a limited recognition of epitopes. This is extremely dangerous for the patient as this indicates that there is not enough BCR or TCR diversity to interact with the array of epitopes required to mount an effective immune response, but also because the epitopes recognized tend to be directed to self (autoimmunity). The resulting clinical profile is immunodeficiency along with autoimmunity. This produces an erythematous rash, often associated with lymphadenopathy, hepatosplenomegaly and other clinical features. Exclusion of TME is crucial for making a diagnosis of Omenn syndrome, as most patients are initially diagnosed with typical SCID, that later develops into Omenn syndrome, thus patients should be monitored for development of Omenn syndrome over time.

Figure 7. Following recognition and binding of the DNA ends, recruitment of proteins FEN, DCLRE1C (also known as Artemis), Tdt and PNKP occurs. DCLRE1C mediates nicking of the hairpin structures at random nucleotides while TdT adds random nucleotides to the 3’ end. This process provides an additional mechanism of introducing variability into the coding regions of the TCR and BCR known as junctional diversity. Created with biorender.com (CA Petersen 2024)

Figure 8. Following end-processing, DNA polymerase µ and ʎ are recruited to fill in single -stranded gaps in the DNA strands. Created with biorender.com (CA Petersen 2024)

Figure 9. following DNA polymerase, a final ligation of the 3’-OH and 5’-P ends is initiated and requires recruitment of proteins XRCC4, XLF (also known as Cernunnos) and DNA ligase IV. *In SCID phenotypes with defects in T and B cells development, but normal NK cell function, genetic mutations in XLF and DNA ligase IV have been identified. Created with biorender.com (CA Petersen 2024)

Figure 10. V(D) recombination is completed when a functional coding joint has be generated. Created with biorender.com (CA Petersen 2024)

Figure 11. SCID with autoimmunity: Omenn’s syndrome. In certain rare forms of SCID with defects in the T and B cell development, but normal NK cell function, the genetic mutations in the affected proteins may permit partial enzymatic activity, known as “leaky” SCID. The restrictive V(D)J recombination permits the development of a limited repertoire of T and B cells which may be directed towards self-antigens leading to an autoimmune response, know as Omenn’s syndrome. Clonal expansion of theses activated T and B cells occurs and blood levels of these cells may appear in range or elevated. Phenotypic examination of the TCR or BCR, however reveals that these cells are oligoclonal in nature. Created with biorender.com (CA Petersen 2024)

Treatment

Current case

The patient was transfused with packed red cells to assist with the severe anaemia. IV immunoglobulin prophylaxis (polygamy) was given during admission. Empiric antibiotic therapy was started at admission as well as four-drug TB treatment initiated.

Bone marrow transplantation

The only cure for SCID is bone marrow transplantation. Extreme care to match the HLA of the donor with the host is the optimal option (usually a living related donor), but an HLA-matched unrelated donor and even HLA partial-mismatched related donor transplantation can be considered with successful outcomes. This approach is successful if the disease is diagnosed within their first 3 months of life and early transplantation before 3.5 months is associated with better overall survival.

Vaccinations

No live vaccines, such as the BCG vaccine, should be administered to patients with SCID prior to bone marrow transplantation.

Intravenous immunoglobulin

The therapeutic function is passive immunization to prevent infection. IVIG can be used to restore antibody levels until the B-cell system is restored with transplantation. However, long-term use fails to change the terminal course of SCID.

With the advances in bone marrow transplantation and gene therapy, patients now have a better likelihood of developing a functional immune system in a previously lethal genetic disease. However, once an infant develops serious infections, intervention is rarely successful

Final Outcome

Unknown. After discharge patient failed to return for follow up and no further hospital admissions have been noted.

References

• Niehues T et al. (2010). More than just SCID–the phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2. Clin Immunol. 2010 May;135(2):183-92. Epub 2010 Feb 20
• Gennery AR et al. (2005). Omenn’s syndrome occurring in patients without mutations in recombination activating genes. Clin Immunol. 2005 Sep;116(3):246-56.
• Notarongelo LD. (2010). Primary immunodeficiencies. J Allergy Clin Immunol. Feb 2010;125 (2 suppl):S182-94
• Dvorak, C. C., Haddad, E., Heimall, J., Dunn, E., Buckley, R. H., Kohn, D. B., Cowan, M. J., Pai, S. Y., Griffith, L. M., Cuvelier, G. D. E., Eissa, H., Shah, A. J., O’Reilly, R. J., Pulsipher, M. A., Wright, N. A. M., Abraham, R. S., Satter, L. F., Notarangelo, L. D., & Puck, J. M. (2023). The diagnosis of severe combined immunodeficiency (SCID): The Primary Immune Deficiency Treatment Consortium (PIDTC) 2022 Definitions. Journal of Allergy and Clinical Immunology, 151(2), 539–546. https://doi.org/10.1016/j.jaci.2022.10.022
• Severe Combined Immunodeficiency (SCID) | NIH: National Institute of Allergy and Infectious Diseases. (n.d.). Retrieved October 26, 2023, from https://www.niaid.nih.gov/diseases-conditions/severe-combined-immunodeficiency-scid
• Slatter, M. A., & Gennery, A. R. (2022). Advances in the treatment of severe combined immunodeficiency. Clinical Immunology, 242, 109084. https://doi.org/10.1016/j.clim.2022.109084

Evaluation – Questions & answers

What is the diagnosis?

What is SCID?

What is the typical history of a child presenting with SCID?

What are the findings on examination of these patients?

What is the pathophysiology of SCID?

How does SCID most commonly occur?

Does SCID always result in a complete primary immunodeficiency where both B and T cells are completely non-functional?

How is new born screening used to diagnose SCID?

Multiple Choice Questions

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