To our knowledge, this the first multicenter study and analysis of GBA recombinant allele in Thai patients, representing the landscape of GD in Thailand in recent years. In the present cohort, all the cases had pediatric onset. GD3 (44.5%) was the most common phenotype in the Thai population, followed by GD2 (40.7%) and GD1 (14.8%), making neuronopathic GD accounting for the majority of cases. When combining our data to previous reports with available information on types of GD and mutations, it still supported that GD3 was the most prevalent phenotype (41.7%) in Thai patients, followed by GD2 (33.3%), and higher proportion of GD1 (25.0%) (Fig. 1B and Table S2) [16–18].
In the present cohort, 85.2% (23/27) of the patients developed first symptoms at age ≤ 5 years and 74.1% (20/27) at age ≤ 1 year of age, indicating relatively early and severe phenotype of Thai GD as compared to those of Western population. All GD3 patients manifested their first symptoms at age ≤ 3 years and two-thirds at age ≤ 1 year. We did not find Norrbottnian (Swedish) phenotypes in patients with homozygous p.L483P, in our cohort. It was not clear, in this study, which patient had GD3a or GD3b, prior to the ERT. Anecdotally, participating physicians recalled that the clinical phenotypes of their GD3 patients were overlapping between 3a and 3b. Patients with GD3 who did not receive ERT died at very young age while those with comparable onset who had ERT were still living. Delayed diagnosis and treatment with ERT led to inevitable neurological progression and death, as seen in one of our patients (GD-26).
The median age of onset of GD1 and GD3 was at 72 and 12 months, respectively, indicating earlier age of onset as compared to westerns affected population. As for, GD2, the onset of symptom was at 4.0 months and natural course did not seem to be different from those described in the literature [1]. The average time to diagnosis was shortest in GD2, at 5.4 months, and longer in GD3 at 19.6 months., and GD1 at 45.3 months. This likely reflected the nature of rapid disease progression of GD2, demanding for immediate investigation.
The p.L483P was the most prevalent allele found in this study, at 66% (33/50 alleles), followed by IVS2 + 1G > A (8%), Rec1a (4%), and IVS6-1G > C (4%) (Table 2). These four alleles together accounted for 82% (41/50) of the mutant alleles identified in this cohort or 75% (51/68) of total Thai GD alleles (Fig. S2) [16–18].
Rec1a allele was found in two patients with neuronopathic GD in the present study, supporting its association with severe disease. Rec1a has been described to be associated with poor clinical outcomes and sometimes mistaken as L444P [6, 7]. Previous studies have shown that a significant number of GD patients were initially assigned with point mutation(s), but in fact they carried recombinant allele(s) [5–8]. All other variants were found, each in one patient. The alleles associated with GD2 in the present study included p.R159W, p.D488H, p.L483P, Rec1a, IVS2 + 1G > A, IVS6-1G > C, IVS7 + 1G > A, and IVS9-3C > G.
In this cohort, the most common genotype was homozygous p.L483P, found in 11 of 12 individuals with GD3, suggesting more homogeneous population of GD3 in Thai affected individuals. The second common genotype was genetic compound between p.L483P and other allele which was found in 11 patients: 1 GD3, 6 GD2, 1 neonatal GD, and 3 GD1. We observed that genetic compounds between p.L483P and the recombinant allele, splicing variants, or known severe missense alleles (p.D488H and p.R159W) would lead to neuronopathic GD while the genetic compounds between p.L483P and p.N409S or p.P305A would result in GD1 with slow clinical progression, as described previously [19]
Twenty-four percent (6 of 25 studied) of patients in this cohort were reassigned with validated genotypes, most of whom (4 of 6) were patients with GD2. Therefore, we suggest inclusion of recombinant allele analysis for Thai GD patients especially those with suspected GD2 and GD3 with rapid progression. Plausible explanations for the discrepancy between the results from the re-genotyping and the prior mutations were using different detailed method of mutation screening, such as using vs not using long ranged PCR, excluding vs not excluding the pseudogene, and limited mutation screening (only for common mutations). Our data support previous observation that the detection of recombinant allele and correct variants of GBA depends on the methodology of genetic analysis [11]. The correct identification of mutant alleles could lead to accurate genetic counseling, reproductive planning, and therapeutic decision making.
Given the [p.S384F + p.W533*] being compounded with p.L483P, resulted in an extreme phenotype of neonatal GD, we hypothesized that the p.S384F could potentiate the deleterious effect of the nonsense mutation, p.W533*, and/or vice versa. Further investigations are required to confirm the pathogenic mechanism of these intriguing alleles. The presence of two alleles in cis has been shown associated with a more severe phenotype in GD, homozygous D409H leading to GD3 with cardiac phenotype in most cases whilst homozygous [D409H + H255Q] resulting in GD2 [20].
Our result suggested that mutation spectrum of GBA in Thai patients was more homogeneous than ever believed and was different from those seen in Caucasian populations, and more similar to those of East Asian descendants [3, 5, 10, 12, 16–18, 21].
The older report of GD in Thailand, during 1966–1998, consisting of 25 GD patients diagnosed over 33 year-period or 0.8 case per year [22], When combining our data with other recent reports of Thai GD, a total number of patients was 33 (27 + 5 + 1) over the 9 years of study (2010–2018) or 3.7 new cases per year [16, 18]. These data could reflect the better awareness of GD among Thai physicians, resource availability including clinical geneticist, diagnostic facilities, and reimbursement for ERT (since 2013) and HSCT (since 2015), which have been escalating lately. We estimate the prevalence of GD in the Thai population at least 1 in 195,426 and carrier frequency of 1 in 221 as ascertained by using Hardy-Weinberg equation and 6,449,073 live births during the study years (http://statbbi.nso.go.th/staticreport). We believe that these numbers could be rather underestimated due to some hospitals not included in the study and patients escaping diagnosis especially those with later onset and mild GD (possible GD1).
As it is known that ERT does not cross blood brain barrier and that patients with GD3 could exhibit more advanced neurological manifestation some time in their life; therefore, new therapeutic approach and/or adjunct treatment targeting to protect neurological involvement may be required to overcome these challenges. Long-term outcome of GD3 with quite homogeneous genotype of homozygous p.L483P deserves further investigation..
There are a number of limitations of this work including missing data due to the natures of the retrospective study and varying experiences of clinicians in detecting specific subtle signs (i.e. oculomotor apraxia) which may perturb the accuracy of clinical data collected and the assignment of GD phenotype in particular GD1 and GD3. Lastly, analysis for reciprocal recombinant alleles and some gene conversion alleles was not included in this study, which might affect the variant distribution, though the effect was believed to be small due to its rarity.