Novel FERMT3 and PTPRQ Mutations Associated with Leukocyte Adhesion Deficiency-III and Sensorineural Hearing Loss

Abstract

Leukocyte adhesion deficiency-III (LAD-III) is a rare genetic disease caused by defective integrin activation in hematopoietic cells due to mutations in the FERMT3 gene. The PTPRQ gene encodes the protein tyrosine phosphatase receptor Q and is essential for the normal maturation and function of hair bundle in the cochlea. Homozygous PTPRQ mutations impair the stereocilia in hair cells which lead to nonsyndromic sensorineural hearing loss (SNHL) with vestibular dysfunction. Here, we report two novel pathogenic homozygous mutations found in two genes, FERMT3 and PTPRQ , in a Brazilian patient with LAD-III and SNHL, which may develop our understanding of the phenotype–genotype correlation and prognosis of patients with these rare diseases.

Introduction

Leukocyte adhesion deficiency-III (LAD-III) is a rare autosomal recessive disease associated with a severe leukocyte adhesion defect leading to recurrent infection and poor platelet aggregation, resembling the Glanzmann-type thrombocytopathy. Additionally, patients may also present with an osteopetrosis-like bone defect. ^{1 2 3 4} Approximately 40 LAD-III patients have been reported. ^{5 6} The single recommended curative therapy is hematopoietic stem cell transplantation (HSCT) which need to be done early in the course of the disease. ^{3 7}

Background

Patients present with recurrent infections associated with leukocytosis (usually bacterial infections, manifesting as early as in the neonatal period), bleeding symptoms (often severe mucosal bleeding, spontaneous bruising, petechial rash, and extensive purpura) due to thrombocytopathy which resembles Glanzmann's thrombasthenia. ^{3 5} Delayed umbilical cord separation may also be present. ⁵ Osteopetrosis-like bone defects have also been cited but not as consistently as the above-described clinical manifestations.

Diagnosis may be suspected based on clinical findings, complete blood counts (leukocytosis), and platelet aggregation assays which demonstrate impaired platelet function. The final diagnosis is made by genetic sequencing of the FERMT3 gene which encodes a protein that acts on hematopoietic cells. ⁵

Management should prioritize early diagnosis of infections as the introduction of antibiotic therapy is imperative for the clinical treatment of patients with LAD-III. Bleeding should be controlled with local hemostatic measures and blood transfusions. Platelet transfusions are required to stop bleeding and red cell transfusions should be used to correct anemia. Recombinant factor VIIa may be helpful, mainly in alloimunized individuals. HSCT is the only curative therapy, although it may bring several morbidities. There is a risk of failure of engraftment that is reduced when the transplant is done early in life. ^{3 6}

Children with LAD-III will usually have poor prognosis and die during childhood due to infection and/or uncontrolled bleeding. The mortality rate for patients who do not undergo HSCT is higher than 75% by 2 years of age. ⁷ HSCT may improve life expectancy and quality of life, with one author reporting on a child living without bleeding manifestations or infections 3 years after transplantation. ³ A recent retrospective multicenter study reported a 3-year overall survival estimate rate of 75% for 11 patients with LAD-III who received an allo-HSCT. ⁷

Hearing loss is one of the most common sensory disabilities in humans. Genetic mutations are the major etiology of sensorineural hearing loss (SNHL). Recent findings reveal that approximately 80% of nonsyndromic genetic hearing loss derives mostly from autosomal recessive mutations, including the mutation in the PTPRQ gene. Hearing loss levels and progression associated to mutations in the PTPRQ gene vary; a relationship between genotypes and phenotypes is still unclear. ⁸

SNHL due to PTPRQ gene mutations manifests with different degrees of hearing loss, with variable progression patterns and can be perceived in the early years (resulting in failure in the newborn hearing screening) or later in life. ⁸

The diagnosis of SNHL can be made by audiogram which shows hearing loss without air-bone gap. Hearing loss levels may vary (from mild to profound losses). Mutations in the PTPRQ gene, which is responsible for encoding a stereociliar membrane protein, are identified by gene sequencing. Temporal bone image (e.g., computed tomography) does not exhibit inner or middle ear malformation. ⁸

SNHL should be managed by a multidisciplinary team that should include a pediatrician, otolaryngologist, phonoaudiologist, and hearing aid specialist. Hearing aids and cochlear implants may improve hearing loss in some cases but it must be individually evaluated.

SNHL due to mutations in the PTPRQ gene is rare and hearing loss levels and its' progression may vary among different individuals, not necessarily with complete deterioration of hearing.

Case Presentation

Clinical Description

The female proband was born at 37 weeks' gestation as the only child of healthy Brazilian first cousins of African origin. The parents are originally from a small village in the Southwest region of Brazil where consanguineous marriages are common. She had an average birth weight, length, and occipital frontal circumference without delay in the fall of the umbilical cord and her developmental milestone was appropriate for age

At the age of 9 months, she was admitted to our service with septic shock history in the neonatal period, recurrent bacterial infections (pneumonias and bloodstream infections), splenomegaly, eczema and persistent leukocytosis with neutrophil predominance (range: 10,000–15,000 neutrophils). At the age of 17 months, she started to have almost daily frequent hemorrhagic episodes, mainly gingival bleeding. At 3 years of age, she presented with partial hearing loss and by the age of 9 years, she was diagnosed with complete deafness and around the same time, she had three spontaneous fractures of the left tibia.

Laboratory tests while she had no bleeding or infection showed hemoglobin: 8.1 g/dL; white blood count 34,720 cells/mm ³ (11,930 cells/mm ³ neutrophils); serum immunoglobulin G (IgG) 2.53 g/L (normal range: 4.52–8.56 g/L); and platelet aggregation was absent in the presence of ADP, arachidonic acid, epinephrine, collagen and normal with ristocetin. Her flow cytometry showed dysfunction of the glycoprotein complex (GPIIb–IIIa complex) with normal protein expression (GPIIb: 96%/GPIIIa: 96%) and her karyotype was 46, XX.

The audiometry showed total bilateral deafness, with no difference between the air and the bone conduction, indicating SNHL.

Molecular Genetic Testing

DNA was extracted from peripheral blood according to standard procedures. Genomic array was performed using HumanCytoSNP-12 BeadChip (Illumina, San Diego, California, United States) and revealed an interstitial deletion of 190 kb on locus 11p15.4, encompassing variants of uncertain significance (VUS) associated with β-thalassemia.

The raw data were extracted using BlueFuse Multi Software (Illumina, San Diego, California, United States). The data were normalized, and the intensity of the sample was divided by the mean intensity across the reference sample to calculate log2 ratios. The SNP and bead arrays supply the B-allele frequency (BAF) which represents the proportion of B-alleles in the genotype. The results were analyzed according to the American College of Medical Genetics guidelines using Database of Genomic Variants (DGV); the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER); and the UCSC Genome Bioinformatics database. Genomic positions are reported according to their mapping on the GRCh37/hg19 genome build.

Fifteen genomic regions with regions of homozygosity were found on several different chromosomes, including relevant regions on short arms of chromosomes 11 and 12. The genomic region at 11q12.1q13.1 (57,218,985–64,212,852) involves the FERMT3 gene, associated with the autosomal recessive LAD-III syndrome.

Subsequently, the whole exome sequencing (WES) using the SureSelect Human All Exon V6 kit (Agilent Technologies, Santa Clara, California, United States) was performed to investigate the genes in regions showing regions of homozygosity according to the array. The generated library was sequenced on an Illumina platform (Illumina, San Diego, California, United States) according to the manufacturer's recommended protocol and the WES kit was able to provide high coverage of exonic regions (up to 60M bp).

The obtained reads were aligned to the human genome reference (UCSC GRCh37/hg19) using the Burrows–Wheeler Aligner (BWA) ⁹ and variants identified with the Genome Analysis Tool Kit (GATK). ¹⁰ After variant filtering, in silico prediction of pathogenicity of variants was performed using: SIFT, ¹¹ PolyPhen-2, ¹² Mutation Taster, ¹³ ExAC Browser, ¹⁴ ClinVar, ¹⁵ dbSNP138, and 1000 Genome Project. ¹⁶ Variants were classified according to the American College of Medical Genetics and Genomics guideline ¹⁶ and OMIM database. These databases were also searched for information that allowed us to compare the expected phenotypes with the clinical features of the patient.

WES data filtering showed two potentially pathogenic variants: a homozygous variant in FERMT3 (c.1825–1G > A), genomic position Chr11:63990784, and other homozygous variant in PTPRQ (c.2792_2793insA) genomic position Chr12:80935488.

Both variants found in this study are likely disease-causing variants. The variant c.1825–1G > A in the FERMT3 gene, identified in homozygosis in this patient, locates in a fully conserved functional domain, possibly affecting RNA splicing and the protein product.

Although this variant has not been described in individuals with FERMT3 -related disease, FERMT3 homozygous pathogenic mutations are associated with leukocyte adhesion deficiency type 3 (MIM no.: 612840), characterized by immunodeficiency manifestations alike to leukocyte adhesion deficiency type 1 (MIM no.: 116920) and bleeding changes similar to that seen in Glanzmann's thrombasthenia (MIM no.: 273800) which is similar to what we observed in this patient.

The variant c.2792_2793insA (p.Leu931fs) in the PTPRQ gene, which was detected in homozygosis in this patient, inserts a base in codon 931, thus changing the reading frame and most likely generating a dysfunctional protein product. Pathogenic homozygous variants in the PTPRQ gene have been associated with autosomal recessive hearing loss type 84A (MIM no.: 613391), who has prelingual progressive sensorineural hearing loss and vestibular dysfunction.

None of these variants have been previously reported in publicly available databases.

Discussion

Here, we documented a patient with LAD-III who had two novel homozygous mutations found in two different genes, namely, FERMT3 and PTPRQ .

The patient was diagnosed with LAD-III due to recurrent bacterial infections, eczema, persistent leukocytosis (neutrophil predominance), and hemorrhagic episodes. The disease is caused by mutations in the FERMT3 gene (located on chromosome 11q12). The FERMT3 gene is responsible for encoding a protein that acts on hematopoietic cells, more specifically the protein interacts with the integrins which are transmembrane adhesion receptors of leukocytes and platelets. The integrins must be activated (after “inside-out” signaling) to improve their binding affinity to their ligands. ^{17 18 19} (MIM no.: 612840; MIM * 607901). This activation process is compromised in patients with LAD-III, ²⁰ resulting in impaired adhesive functions of integrins on leukocytes and platelets (MIM * 607901). The signaling defect affects β3 integrin fibrinogen receptor αIIbβ3 (glycoprotein complex GPIIb–IIIa) which explains the Glanzmann thrombasthenia-like laboratory findings. ^{5 21 22}

Integrins are expressed in hematopoietic cells, including T- and B-lymphocytes, acting as an essential component for B-cell adhesion to lymph nodes. Thus, dysfunctional integrins may lead to innate and adaptive immune dysfunctions. There are few reports showing defects in adaptive immunity in LAD-III causing hypogammaglobulinemia. ^{23 24} Our patient required regular intravenous immunoglobulin replacement therapy due to hypogammaglobulinemia. The patient's FERMT3 variant might be associated to IgG deficiency. ^{25 26 27} To the best of our knowledge, this mutation has not been previously described.

The integrins are also important for osteoclasts adhesion and osteoclast-mediated bone resorption. This impaired bone resorption may explain the osteopetrosis-like manifestations (spontaneous fractures) in our patient. ²⁵

The patient's deafness might have been explained by a conductive component, due to impaired bone resorption. ²⁸ Unexpectedly, the patient presented only with SNHL.

The PTPRQ gene is responsible for encoding tyrosine phosphatase receptor Q. The hair bundle in the cochlea requires this receptor for normal maturation and function. Homozygous mutations of the PTPRQ gene are associated with nonsyndromic SNHL and vestibular dysfunction due to defects of stereocilia in hair cell. So far, 10 pathogenic mutations in the PTPRQ gene associated to SNHL have been reported. ⁸ Our patient's variant c.2792_2793insA (pLeu931fs) causes a frameshift and most likely generates a dysfunctional protein. Thus, it is likely that the variant detected in our patient, and which has not been previously described, is a new pathogenic mutation.

Previously, LAD-III was reported in Turkish, Arab, Maltese, and African American individuals, with reports revealing distinct mutations in the FERMT3 gene. ^{5 29} PTPRQ gene mutations were described in individuals of Dutch and Moroccan origins. The present case is the first Brazilian patient carrying previously unreported mutations in two different genes. The presence of both mutations in a single individual may be explained by high consanguinity observed in this family.

Conclusion

In conclusion, the further reports of new mutations in both FERMT3 and PTPRQ genes allow us to better understand the phenotype–genotype correlation and the prognosis of patients with these rare diseases.

Funding Statement

Funding None.