A founder RAB27A variant causes Griscelli syndrome type 2 with phenotypic heterogeneity in Qatari families

Griscelli syndrome type 2 (GS2) is a rare autosomal recessive disorder caused by pathogenic variants in the RAB27A gene and characterized by partial albinism, immunodeficiency, and occasional hematological and neurological involvement. We reviewed and analyzed the medical records of 12 individuals with GS2 from six families belonging to a highly consanguineous Qatari tribe and with a recurrent pathogenic variant in the RAB27A gene (NM_004580.4: c.244C > T, p.Arg82Cys). Detailed demographic, clinical, and molecular data were collected. Cutaneous manifestations were the most common presentation (42%), followed by neurological abnormalities (33%) and immunodeficiency (25%). The most severe manifestation was HLH (33%). Among the 12 patients, three patients (25%) underwent HSCT, and four (33%) died. The cause of death in all four patients was deemed HLH, providing evidence for this complication's fatal nature. Interestingly, two affected patients (16%) were asymptomatic. This report highlights the broad spectrum of clinical presentations of GS2 associated with a founder variant in the RAB27A gene (c.244C > T, p.Arg82Cys). Early suspicion of GS2 among Qatari patients with cutaneous manifestations, neurological findings, immunodeficiency, and HLH would shorten the diagnostic odyssey, guide early and appropriate treatment, and prevent fatal outcomes.

Three distinct types of GS have been described based on molecular analysis. GS type 1 (GS1) (OMIM: 214450) is caused by pathogenic variants in the MYO5A gene and presents with hypopigmentation and primary neurological deficit with an intact immune system (Pastural et al., 1997). GS type 2 (GS2) (OMIM: 607624) is caused by pathogenic variants in the RAB27A gene and manifests with partial albinism that can be subtle and immunodeficiency that mainly affects T lymphocytes and natural killer (NK) cells, with complications ranging from frequent pyogenic infections of the skin and internal organs to HLH. Neurological impairment, which is a well-recognized feature of GS1 at birth, is increasingly reported in GS2 as a spectrum of neuropathology that develops at a later stage after birth and includes seizures, strabismus, nystagmus, ataxia, hemiparesis, facial palsy, and impairment of cognitive functions (Anikster et al., 2002;Rajadhyax et al., 2007). GS type 3 (GS3) (OMIM: 609227) is caused by pathogenic variants in the MELANOPHILIN gene and is characterized by unusually light skin and silvery-gray hair. Unlike the other forms of GS, the central nervous system (CNS) and the immune system are not involved in GS3 (Ménasché et al., 2003).
In GS2, the RAB27A gene that encodes a GTPase called Rab27a is disrupted and affects a melanosome-anchoring complex in melanocytes as well as exocytosis of cytolytic granules from T lymphocytes and NK cells, thus increasing the susceptibility to recurrent infections and culminating in life-threatening HLH (Bahadoran et al., 2003;Marsh, 2018;Zhang et al., 2016). Ashen mice with a loss of function variant in the RAB27A gene provide a mouse model for GS and demonstrate defects in melanosomes, which abnormally cluster in the perinuclear region of melanocytes, and lytic granules within cytotoxic T lymphocytes (CTLs), which prevents CTLs from killing target cells (Barral et al., 2002;Haddad et al., 2001;Hume et al., 2001;Stinchcombe et al., 2001;Wilson et al., 2000;Wu et al., 2001). The Rab27a protein, a member of the Rab family, has been identified in different cell types, including oligodendrocytes, as a potential positive regulator of exosome release and proteolipid trafficking (Shen et al., 2016). Meeths et al. (2010) suggested that GS's neurological abnormalities could be associated with the inflammatory HLH state and lymphohistiocytic infiltration of the brain rather than a result of the Rab27a deficiency itself. However, another theory proposed that neurological abnormalities could be part of the underlying disease, directly related to the defective Rab27a protein, and occurring in the absence of immunodeficiency (Panigrahi et al., 2015) .

| OBJECTIVE
This case series aims to describe the broad spectrum of clinical presentations of GS2 in Qatari families in association with a recurring RAB27A pathogenic variant (NM_004580.4: c.244C > T, p.Arg82Cys) ranging from asymptomatic healthy individuals to patients with isolated dermatosis and skin manifestations, to patients with more severe neurological and immunological complications, including fatal HLH. In this article, we refer to this RAB27A variant as p.R82C.

| MATERIALS AND METHODS
In this case series, we report on 12 individuals from six Qatari families diagnosed with GS2 at the Medical Genetics Department at Hamad Medical Corporation, Doha, Qatar, between 2002 and 2019. We conducted a thorough review of patient medical records and collected detailed demographic and clinical data: age at presentation, age at diagnosis, gender, family history, clinical presentation, diagnostic workup (hematological, immunological, neuroimaging, and molecular genetic), management, and disease outcome. Medical data were stratified based on the most common systems involved, including neurological (e.g., seizures, strabismus, nystagmus, hemiparesis, developmental delay, intellectual disability, and abnormal brain imaging), hematological (e.g., HLH), dermatological, and immunological.
Clinical exome sequencing (CES) (XomeDx) was performed on all probands at GeneDx, USA. DNA was extracted from submitted specimens, enriched for most genes of the human genome using a proprietary capture system developed by GeneDx for next-generation sequencing with copy number variant (CNV) calling (NGS-CNV), and sequenced with paired-end reads on an Illumina platform (GeneDx, n. d.). Bi-directional sequence reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSC hg19 (GeneDx, n.d.). A custom-developed analysis tool (XomeAnalyzer) was used to filter and analyze the data to identify the sequence and copy number variants (Retterer et al., 2016

| Family A
The index patient A-1 is a 13-year-old female. She was the product of a full-term normal vaginal delivery with a birth weight of 3,200 g, a length of 51 cm, and a head circumference of 35 cm ( Figure 1a). Apgar score was 9 at 1 min and 10 at 5 min. She was diagnosed with classical homocystinuria through the Qatar National Expanded Metabolic Newborn Screening Program and was immediately started on treatment with a good outcome. Her parents are first cousins, and she has an older brother, all reported to be healthy.
At the age of 1 year, the patient started to develop a progressive skin rash (eczematous patches/granulomatous), mainly on the face and limbs. Different dermatologists evaluated her over several years without reaching a final diagnosis. At the age of 12 years, CES was performed and showed a homozygous pathogenic variant p.R82C in the RAB27A gene. Interestingly, trio analysis of the exome sequencing data revealed that the mother A-2, who is asymptomatic, was F I G U R E 1 Family Pedigree (A-F) homozygous for the same RAB27A pathogenic variant, while the father and brother were heterozygous.

| Family B
The index patient B-1 is a previously healthy 4-year-old male who presented with a non-paralytic squint, imbalanced gait, slurred speech for 2 weeks, abnormal brain magnetic resonance imaging (MRI), and three hypopigmented macules on the trunk (Figure 1b). Subsequently, his condition acutely deteriorated with progressive encephalopathy, impaired consciousness, peripheral hypotonia, bilateral facial palsy and weakness, and bulbar palsy. The patient was the product of a fullterm normal vaginal delivery with a birth weight of 3,120 g, a length of 50 cm, and a head circumference of 33 cm. Apgar score was 9 at 1 min and 10 at 5 min. His parents are first cousins, and he has a younger brother B-2 who is apparently healthy and developing normally. There is a positive family history of hypopigmentation in a few family members.

Initial investigations included an electroencephalogram (EEG) that
showed slow background but no specific epileptic findings and an initial metabolic screen that revealed normal lactate, ammonia, and amino acids. All testing for bacterial and viral pathogens, as well as immunological workup and cerebrospinal fluid (CSF) analysis, were negative. Urine organic acids and CSF neurotransmitters were also normal. A repeated brain MRI showed bilateral symmetrical basal ganglia high signal with adjacent cortical and subcortical involvement.
A provisional diagnosis of biotin-thiamine-responsive basal ganglia disease was then proposed, and the patient was started empirically on biotin and thiamine. However, there was no improvement, and molecular genetic testing of the SLC19A3 gene was negative. He was also started on methylprednisolone because of the initial concern about a possible immune disorder. CES was performed and showed a homozygous pathogenic variant p.R82C in the RAB27A gene. Interestingly, his asymptomatic brother B-2 was also found to be homozygous for the same RAB27A pathogenic variant. B-1 underwent HSCT without significant complications. He has no neurological complications posttransplant. B-2 underwent preemptive HSCT. It was paternally derived bone marrow transplant with subsequent graft failure and poor engraftment. Thereafter, he received two DLI (Donor lymphocyte infusion). However, he remained neurologically stable.

| Family C
The index patient C-1 is a 7-year-old male with a seizure disorder, global developmental delay, white matter abnormalities, and fair skin and hair compared to his parents and siblings ( Figure 1c). Fetal life was complicated by gestational diabetes, intrauterine growth restriction (IUGR), oligohydramnios, and positive group B streptococcus. The patient was the product of a cesarean section for fetal distress with a birth weight of 2,200 g, a length of 46 cm, and a head circumference of 31 cm (all parameters at the 10th percentile). He was admitted to the neonatal intensive care unit (NICU) for 4 days for suspected sepsis, resolving thrombocytopenia and hyperbilirubinemia, and a brief jerky movement of the left upper limb. He was re-admitted at the age of 10 days with a history of abnormal jerky movements of the left upper and lower limbs associated with facial twisting, and he was treated with phenobarbitone. His parents are first cousins, he has a younger sister with right hemiparesis and MRI finding of leukodystrophy, and he has a paternal uncle with intellectual disability.
On physical exam, there was spasticity with scissoring gait, brisk reflexes, and sustained clonus. Besides, three hypopigmented macules were noted. The patient had an extensive workup including lactate, ammonia, plasma amino acids, urine organic acids, urine for purines and pyrimidines, congenital disorders of glycosylation (CDG) and peroxisomal studies, and chromosomal microarray analysis, which were all normal. Brain MRI revealed significant interval resolution of intraparenchymal hemorrhage with residual hemosiderin deposition in the right high parietal and left occipital lobes, diffuse white matter volume loss with periventricular and centrum semi-ovale representing hypoxic/ischemic changes, diffuse thinning of the corpus callosum, and mild dilatation of ventricular system. Magnetic resonance spectroscopy showed a lactate peak and areas of mildly elevated choline.

CES was done and revealed a homozygous pathogenic variant p.R82C
in the RAB27A gene.

| Family D
The index patient D-4 is a 19-year-old male with no previous remarkable medical history diagnosed with primary immunodeficiency with NK cell dysfunction at the age of 16 years ( Figure 1d). His parents are consanguineous, and there is an extensive family history of primary immunodeficiency and HLH. His sibling, D-5, is a 17-year-old male diagnosed with primary immunodeficiency with NK dysfunction at the age of 14 years. Two older siblings D-2 and D-3 were diagnosed with familial HLH and passed away at the ages of 11 and 12 years, respectively. D-2 presented at the age of 10 years with acute encephalitis, neutropenia, and thrombocytopenia. At the age of 11 years, he developed a progressive course of pancytopenia, multiple intracranial hemorrhagic lesions, intractable seizures, demyelinating sensorimotor neuropathy, and viral infection. He died of HLH. D-3 presented at the age of 9 years with lateral gaze nystagmus, ataxic gait, generalized hypotonia, bilateral tremors, and head titubation with abnormal brain MRI. Three café au lait spots (1 × 1 cm each) were noted on her right leg during the physical exam. She later developed a viral infection and died of HLH.
The mother D-1 was also diagnosed with HLH with a prior Epstein-Barr virus (EBV) infection and died at the age of 39 years.
CES performed on this family revealed that D-1, D-3, D-4, and D-5 are homozygous for a pathogenic variant p.R82C in the RAB27A gene. D-1 was diagnosed with GS2 postmortem using previously banked DNA. The father and the three remaining siblings were found to be heterozygous for the familial pathogenic RAB27A variant p.R82C in the gene and remain asymptomatic. This family was previously published (Netter et al., 2016).

| Family E
The index patient E-1 is a 22-year-old male with no previous remarkable medical history. He has no significant family history besides consanguineous parents (Figure 1e). He was admitted with generalized weakness, night sweats, and weight loss of 15 kg in 6 months. On examination, he was found to have small palpable lymph nodes. Laboratory investigations showed pancytopenia and a picture of disseminated intravascular coagulopathy (DIC). Pan computed tomography (CT) revealed sub-centimetric cervical lymph node, intra-abdominal enlarged lymph nodes, and hepatosplenomegaly. Bone marrow examination suggested hypercellular with trilineage hemopoiesis and atypical lymphoid infiltrate. The patient traveled abroad and lost follow-up.
Four months later, he was admitted to the intensive care unit (ICU) with a decreased level of consciousness. MRI of the brain showed demyelination features, and lumbar puncture (LP) was unremarkable.
A second bone marrow exam showed a picture of significant hemophagocytic activity in addition to increasing triglyceride and ferritin levels, which led to a diagnosis of HLH. He was started on HLH protocol; however, pancytopenia worsened daily with a picture of DIC and multiple organ failure. He was kept on supportive transfusions until he died at the age of 22 years. CES was performed and revealed a homozygous pathogenic variant p.R82C in the RAB27A gene.

| Family F
The index patient F-1 is a six-year-old male (Figure 1f). He was the product of a full-term normal vaginal delivery with a birth weight of 3,000 g. He was admitted to the NICU for 1 week after birth due to fever to rule out sepsis. At the age of 2 months, he started to have eczema. Recurrent cough and breathing difficulties led to a diagnosis of reactive airway disease (RAD) with findings on chest X-ray. At the age of 12 months, he was admitted to the pediatric intensive care unit (PICU) due to fever, respiratory distress, abdominal distention, decreased activity, and decreased oral intake. He was found to have bronchiolitis due to respiratory syncytial virus (RSV) and past infection with both EBV and cytomegalovirus (CMV). Neck ultrasound (US) and abdomen US revealed generalized lymphadenopathy, significant hepatosplenomegaly, and umbilical hernia. He was evaluated by different specialties to rule out various etiologies for his symptoms, but to one family (family D). The gender distribution was unequal, with eight males (66%). While the age at presentation ranged from less than 1 year to 21 years with a median of 9.5 years of age, we found the age at diagnosis to differ significantly, ranging from 3 to 42 years with a median of 11.5 years of age. Consanguinity was present in 100% of the patients. All families were found to belong to the same Qatari tribe. There was a positive family history for 10 out of 12 (83%) patients (Table 1).

| Clinical findings and outcomes
The most common clinical manifestations found in patients were the cutaneous ones that were identified in 5 out of 12 patients (42%): T A B L E 1 Summary of demographic and clinical characteristics of the 12 Qatari patients with GS2 four out of five patients (80%) had albinism, and one out of five patients (20%) had hypopigmentation. The second most frequent manifestations were the neurological abnormalities in 4 out of 12 patients (33%), all of which had an abnormal brain MRI (100%).
Among the four patients with neurological involvement, two had seizures (50%), two had strabismus (50%), two had hemiparesis (50%), two had a developmental delay (50%), and two had an intellectual disability (50%). The most severe manifestation identified in our patients was HLH, which was found in four individuals (33%), three of which belong to the same family (family D). Most (75%) of the patients who had HLH had a viral infection before HLH: one had EBV, and two had CMV. The immunodeficiency was also found in three patients (25%), two of which belong to one family (family D  (Aksu et al., 2003;Ashrafi et al., 2006;Harfi et al., 1992;Mamishi et al., 2008;Masri et al., 2008;Mishra et al., 2014;Ohbayashi et al., 2010;Patiroglu et al., 2016;Shamsian et al., 2010 (Panigrahi et al., 2015). Alternatively, neurological abnormalities could be secondary to cellular infiltration of the nervous system when HLH develops (Marsh, 2018). There are  no extensive studies delineating the rate or age at diagnosis of primary neurological abnormalities. Moreover, there is a gap in the medical literature regarding studies that examine the association between the specific type of pathogenic variant in GS2 and the occurrence of different types of primary abnormalities. These two facts could be attributed to the rarity of GS2, a disease where most publications consist of case reports of one to two patients or small cohorts (Al-Ahmari et al., 2010;Bindu et al., 2015;Meeths et al., 2010;Rajyalakshmi & Chakrapani, 2016;Sarper et al., 2002). The presentation of progressive unexplainable neurological diseases such as seizures, strabismus, nystagmus, hemiparesis, developmental delay, or intellectual disability in children who were previously healthy should raise suspicion of GS2, regardless of the simultaneous presence of cutaneous or immunological abnormalities. Considering GS2 as a primary differential diagnosis in such cases would enable early detection, prompt initiation of appropriate and effective treatment, and avoid the fatal outcome of HLH.
Although we reported a lower frequency of HLH (33%) in our patients compared to previously reported cases (71%), it is still crucial to consider and suspect GS2 in the above-described pediatric group given the high fatality rate of HLH and the limited availability of HSCT that can improve overall survival. In this case series, almost half of the patients had a viral infection, mainly EBV, and three out of four patients with fatal HLH complications (75%) had a viral infection, 50% of which were EBV-related. HLH-related EBV infection is the most common viral infection in children (Kogawa et al., 2014). Although the pre-transplant viral infection was documented in 42% of our patients, HLH was noticeably less frequent than previously described. Kuskonmaz et al. (2017)  Although partial albinism or hypopigmentation is still considered a characteristic feature of GS2 (Cetica et al., 2015;Netter et al., 2016;Tesi et al., 2018), the p.R82C variant is known to present only with cytotoxic defect, sparing pigmentary dilution (Netter et al., 2016

| CONCLUSIONS
In conclusion, all of the patients with GS2 in our cohort were homozygous for the same founder pathogenic variant p.R82C in the RAB27A gene; albeit striking phenotypic variability was noted among them.
We recommend that GS2 should be considered in the differential diagnosis of acutely deteriorating patients with immune dysregulation, HLH, or CNS involvement, especially among consanguineous Qatari families, to shorten the diagnostic odyssey, guide early and appropriate treatment, and prevent possible fatal outcomes. We also recommend targeted testing of this founder RAB27A variant p.R82C in the identified Qatari tribes to identify asymptomatic carriers and asymptomatic homozygotes who may benefit from preventative measures and genetic counseling in the premarital, preconceptional, or prenatal period.
We acknowledge our partner laboratory GeneDx, USA for running the CES on our patients and for conducting bioinformatics analyses to study the age of the p.R82C variant in our population.

CONFLICT OF INTEREST
There are no commercial associations that might pose a conflict of interest. We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of the authors listed in the manuscript has been approved by all of the authors. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In doing so, we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animals or human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the corresponding author is the sole contact for the editorial process (including the editorial manager and direct communications with the office). He/She is responsible for communicating with the other authors about the progress, submission of revisions, and the final approval of proofs. We confirm that we have provided a current, correct email address that is accessible by the corresponding author.

AUTHOR CONTRIBUTIONS
Reem data acquisition; data analysis; statistical analysis; manuscript preparation; manuscript editing; manuscript review and approval.

ETHICS STATEMENT
The study was approved by the Ethical Committee of the Hamad Medical Corporation, Doha, Qatar (MRC-04-20-325). All clinical investigations were conducted according to the principles expressed in the 1964 Helsinki declaration and its recent amendments.

DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.