Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children

Our study included 34 Egyptian patients with LAD-I phenotype evaluated at PPIU between 2009 and 2023. Informed consents were obtained from parents or legal guardians and the study was approved by the local institutional review board. Details of each patient’s personal medical history, clinical and laboratory findings were recorded, and LAD-I was diagnosed according to the International Union of Immunodeficiencies (IUIS) criteria for IEI [5] and the European Society for Immunodeficiencies (ESID) criteria. Genetic material was available for only 28 patients.

88% of our patients were born to consanguineous families, hence highlighting the burden of autosomal recessive IEI in highly consanguineous populations like Egypt, Pakistan, and Israel [12, 13]. Although a family history of sibs with a typical LAD-I clinical phenotype was reported in 49% of the study population, only 2 of those index cases/sibs presented to be diagnosed as LAD-I before their demise, and this was comparable to other studies [3, 13].

97% of the patients presented in their first year of life (except for P26). The delay in diagnosis almost halved in the last five years, with a median of 2.3 months. This reflects the improved awareness of many developing countries’ pediatricians, as currently witnessed in Egypt and Pakistan [13]. Patient 26 and two of her siblings initially symptomatized around 2 years of age with a mild phenotype (recurrent skin rash and ulcers, yet thrived well); they presented for diagnosis at the ages of 12, 5 and 13 years, respectively. The same family had a sib manifesting and died before the age of 1 year with a stormy course of recurrent infections and sepsis. One of the siblings received a fully matched related HSCT at the age of 9 years, which was unfortunately complicated by GVHD, resulting in his loss. P30 presented for diagnosis at the age of 7 years, although his symptoms started at the age of 1 month. The diagnosis was also reached at an older age in P3, P7, P12, and P25 (at the ages of 3, 5, 3 and 4 years respectively), despite early symptoms in the first few months of life. Celiksoy et al.. described three Turkish siblings with a very similar presentation who were diagnosed with LAD-I in their early adolescence [14].

Umbilical stump disease and/or skin and soft tissue disease were the first presenting symptoms in all patients included in this study, among many other studies [3, 13‐18, 20‐23].

Anecdotally delayed umbilical separation, however, is not a hallmark of the disease, while umbilical stump disease are a similar conclusion reported in other studies [12, 15, 19].

Pneumonia, chronic otitis media, enteropathy, and oral thrush were also common symptoms presenting throughout the disease course [3, 15].

In patients surviving to older ages, autoinflammatory symptoms prevailed in the form of periodontitis and loss of teeth, as well as enteropathy. This was evident in several studies recognising periodontal disease as the most common manifestation in surviving older LAD-I patients without HSCT [24, 25]. The introduction of Ustekinumab for those patients at the same dose used in patients with psoriasis yielded promising outcomes in the resolution of persistant oral inflammation and extensive sacral wounds. Although Ustekinumab is effective in early complete blocking of interleukin-17, prolonged therapy is recommended in patients with periodontitis brought on by the ongoing inflammatory process [26]. Unfortunately, this modality of treatment is not yet conducted in our center due to financial restraints.

The scope of opportunistic, aggressive organisms isolated from our patients did not differ from the ones described in the literature, owing to the chronic inflammatory nature of the skin and soft tissue lesions [15, 18, 27, 28].

As has been reported in similar studies, LAD-I⁰ was also the predominant phenotype in our population [1, 3, 22, 29, 30]. All LAD-I⁰ had severe manifestations early in life, and 65% of the ones following in our clinic lost their lives. Only 3 patients were LAD-I⁻, and their CD18 level was barely above 2% (P28, P29, and P30). All of them are alive, however, suffering from aggressive autoinflammatory symptoms in the form of periodontitis and enteropathy [4, 25, 26].

Anemia in LAD-I patients is a well-documented finding (prevalent as high as 67%, 72%, and 85% of patients in Wolach et al., Yaz et al., as well as our cohort, respectively), which can be attributed to chronic inflammation and poor intake due to recurrent infections and frequent hospitalization [12, 15].

58% of the study population perished. This is sadly comprehensive in our setting, given the severity of the disease and the limited national access to a timely, curative HSCT. Comparable bleak outcomes can be found in a Spanish American study in 2018; an Indian and a Pakistani study in 2020, representing the morbidity and mortality burden associated with LAD-I disease. In Ankara, Bakhtiar et al. reported 3-year survival estimates of 84% in a cohort of 69 LAD-I patients following HSCT. The best survival rates were found in patients with sibling(s), matched family, or unrelated donor transplants. GVHD was the major cause of death, followed by infections [3, 4, 13, 32].

ITGß2 gene is located on chromosome 21q22.3 (OMIM *600,065), where a range of mutations have been identified in patients with LAD-I. A few mutations could cause a nonfunctional but normally expressed CD18 molecule. Most ITGß2 mutations lead to reduced/null expression of β2-integrins on the leukocyte surface [33].

In our study, direct Sanger sequencing of the ITGß2 gene revealed 16 disease-causing variants in 28 patients. These mutations were clustered mostly in exons 4, 6, 7, and 13. The frequency of the variant c.850G > A (p.Gly284Ser) was high in 10/28 patients (36%). This may reflect a founder effect due to high consanguinity and inbreeding in the Egyptian population, as previously reported in variants in other genes such as CYBA c.295_301del causing chronic granulomatous disease (CGD) [34].

About 43% (n = 7) of the variants were located in exon 5–9, a highly conserved region of the extracellular domain of CD18, followed by the cysteine-rich repeat region (CRR) domain (25%, n = 4). Similarly, Roos et al., 2002 stated that most of the single nucleotide variations are found in a ~ 240-residue domain that is highly conserved in all β integrin subunits and encoded by exons 5–9 of ITGß2 [35].

Out of the 16 identified disease-causing variants; 6 were previously reported, while the remaining 10 were novel and were not found in ExAC nor1000G suggesting heterogeneity in the mutation spectrum for LAD-I.

Two novel variants were detected in Exon 6 of the ITGß2 gene: P14 with the c.500G > T variant, causing a change of amino acid sequence at position 167 from glycine to valine. Being 2 nucleotides away from the splice site might cause a splice site change and affect the protein features as this position is highly conserved. While P24 had a c.652 C > T variant causing severely truncated protein after 218 amino acids, the loss of protein function in this case can be due to nonsense-mediated mRNA decay [36].

Three novel variants were detected in Exon 4 of the ITGß2 gene; the missense variant c.185G > A was identified in P19 and P28, it caused a change of amino acid at position 62 from cysteine to tyrosine, which might lead to changes in the protein features. This variant has not previously been reported in LAD-I patients, although an adjacent missense variant c.184T > C; p.Cys62Arg was previously reported by Van De Vijver et al. in 2012 [37]. P18 showed c.306dupA causing a frameshift in the protein at position 103 from valine to serine and a truncation of the protein at amino acid 141. While P4 showed a homozygous deletion c.307delG causing a premature truncation of the protein at position 103.

Three novel variants were detected in Exon 13 of the ITGß2 gene; The missense variant c.1663G > A found in P12, caused a change of amino acid at position 555 from glycine, which is a neutral nonpolar amino acid to arginine, which is a basic polar amino acid, this is predicted to lead to changes in the protein features. While P8 showed a homozygous missense mutation c.1670G > A, causing a change of amino acid sequence at position 557 from cysteine to tyrosine. Although this variant is not previously reported in LAD-I patients, another missense variant in the same position (c.1670G > C, p.C557S) was previously reported in van de Vijver et al., 2012 [37].

P17 showed a homozygous deletion c.1848delC causing a frameshift in the protein at position 617 from glycine to alanine and a truncation of the protein at amino acid 632. Loss of protein function in this case can either be due to protein truncation or nonsense-mediated mRNA decay.

P11 had a novel nonsense variant in exon 11 c.1377 C > A, causing a truncated protein after 459 amino acids, probably producing truncated mRNA, which was degraded through nonsense-mediated mRNA decay.

P1 showed heterozygous deletion in exon 14 c.2003_2004delAG causing a frameshift in the protein at position 668 from glutamate to glycine and a truncation of the protein at amino acid 690, as well as a heterozygous variant c.850G > A commonly found in our patients.

Four PND tests were offered to three families, and only one proved to have a homozygous pathogenic variant. PND services can enable early postpartum intervention in cases where families decide to proceed with the pregnancy that would result in an affected baby. It is worth mentioning that a similar approach was taken with other IEI disorders genetically diagnosed at our center.

Though awareness of LAD-I diagnosis in Egypt has markedly improved recently, as evidenced by shortening in the delay of diagnosis, the death toll is still too high owing to the limited access to a timely HSCT. Further molecular studies, though still challenging, are essential to better characterize the disease genotype in the region, as more than half the detected mutations in our cohort were novel.