Epidemiology and Genotyping of Patients with Lysosomal Storage Disease in Malaysia.

Background: Lysosomal storage disorders (LSD) are storage disorders involving malfunction of degradation enzymes in lysosome. More than 50 types of LSD have been discovered, which includes the group of mucopolysaccharidoses (MPS), sphingolipidoses, oligosaccharidoses, mucolipidoses, lipoprotein storage disorders, lysosomal transport defects and neuronal ceroid lipofuscinoses and others. The aims of this study were to calculate the birth prevalence and carrier frequency of LSDs in the Malaysian population; to compare our results with reported epidemiologic data from other populations, and to describe the mutation spectrum in Malaysia. From 2008 to 2017, 2.1% (92/4338) suspected patients were diagnosed with LSD. Results: The prevalence of LSD in Malaysia was 1/231,904 live births. The combined prevalence of MPS was 1/292,401 with its subtype of MPS II presented the highest calculated birth prevalence of 1/221,425. Within the group of sphingolipidoses, the combine prevalence was 1/770,777 with Fabry as the most common disorder with calculated prevalence of 1/193,203 followed by metachromatic leukodystrophy (MLD) (1/494,514). MLD is more common among people of Iban ethnicity with the prevalence of 1/6,981. Pompe and mucolipidoses type II are the less common subtypes of LSD with a prevalence of 1/1,694,634 and 1/2,229,516, respectively. Conclusion: Overall, although the prevalence of LSD in Malaysia may be underestimated, the prevalence of MPS is consistent with other reported in East Asian countries. and implementation of preventative programs such as premarital testing and neonatal screening can be proposed to the health authority i.e., Ministry of Health Malaysia. This study described the birth prevalence of LSD and its carrier frequencies in Malaysian population. Our estimated prevalence was compared to four other countries namely Czech Republic, United Arab Emirates, Northern Portugal, and The Netherlands. Furthermore, the unique mutation spectrum among Malaysian population was also being reported.

Data from individuals who had been tested for mucopolysaccharidoses (MPS) screening (Glycosaminoglycans [GAGs] quantitation and characterization) and/or urinary oligosaccharidoses and/or Pompe screening and/or multiplex LSD screening from 2008 to 2017 were included in this retrospective study.

Variables and measurement
Variable measurand in this study includes gender, ethnicity, regional, age at diagnosis, frequency of LSD, LSD group, and LSD type.

Data collection
Laboratory ndings including results interpretation were retrieved from the laboratory records and reviewed. Patients were included in the study only if the diagnosis of LSD was con rmed with both screening and con rmatory testing.

Laboratory processes and tests
All laboratory diagnoses from screening to con rmatory were done in IMR which is the reference centre providing comprehensive tests for LSD. We received samples from all healthcare facilities including private hospitals throughout Malaysia. There were four screening tests for LSD: (i) urinary quantitation of GAGs using spectrophotometry and characterization of GAGs using one-dimension high resolution electrophoresis for determination of MPS subtype; (ii) urinary qualitative oligosaccharidoses by thin layer chromatography (TLC) method for screening of α-mannosidosis, α-fucosidosis, GM 1 gangliosidosis, GM 2 gangliosidosis, β-mannosidosis, Pompe/Glycogen Storage Disease Type II and Schindler disease; (iii) Pompe screening by uorometry method using uorometer for quantitation of α-glucosidase enzyme in dried blood spots (DBS); and (iv) multiplex enzyme assay in DBS using tandem mass spectrometry for screening of Niemann-Pick disease, Fabry disease, Pompe disease, Krabbe disease and Gaucher disease. The con rmatory diagnoses of LSD were either by enzyme assay for the absence or reduction of enzyme activity in leukocytes or plasma, or by mutational analysis of respective genes. PCR and bidirectional sequencing were applied to identify mutations and were carried out at accredited genetic laboratory.

Data Analysis
Disease birth prevalence was expressed as number of patients per 100,000 live births and calculated using the method reported by Poorthuis et al. [2]. Brie y, the prevalence was set as the total number of Malaysian patients with the speci c disease divided by the total number of Malaysian live births during the birth period.
The birth period was de ned as the time interval between year of birth of the oldest patient and year of birth of the youngest patient. Live births per year were obtained from the Department of Statistics Malaysia (https://www.dosm.gov.my). We used Pinto et al. [1] method to estimate disease birth prevalence when only a single patient was diagnosed with the disease, using the number of live births between 1974 and 2017 [1]. The overall prevalence was calculated from the total prevalence of each LSD phenotype.
Comparisons between countries were evaluated by selecting four countries which are United Arab Emirates representing Asian population; while Czech Republic, The Netherlands, and Northern Portugal representing Caucasian population. These countries were chosen due to its high frequency of LSD cases within its population.
As for spectrum mutation analysis, the sequencing results obtained were evaluated by aligning with human reference sequence retrieved from GenBank® (http://www.ncbi.nlm.nih.gov) database; and the pathogenic and likely pathogenic variants selection were performed by comparing with available information from publication as well as clinical and population frequency databases. Carrier frequency was calculated using the Hardy-Weinberg equation (Strachan and Read 2004) online calculator at http://perinatology.com/calculators/Hardy-Weinberg.htm Descriptive statistics were presented as number and percentage for categorical variables. Patients with missing gender and age data were excluded from descriptive statistics. Median with interquartile range (IQR) were used for data that were not normally distributed. Data collected was tabulated using IBM

Patient demographics
Demographics of Malaysian patients is presented in Table 1. Out of 4338 patients who were clinically suspected of having LSD, 92 (2.1%) were diagnosed with 15 different subtypes. The gender distribution was almost balance between males and females with 55.4% and 44.6%, respectively. Most patients (93.6%) were below the age of 18 years at diagnosis. Individuals at or over the age of 18 years were considered as adults. Nearly 50% of adult cases were diagnosed with adult form of MPS IV. Most of our patients (44.6%) were originated from central region. In general, the age median at diagnosis was 2.54 years, ranging from 0.01 (4 days) to 40 years. Comparing to this, MPS Type IVA patients were diagnosed 7 years late whereas MPS Type I patients were diagnosed as early as 1 year of age.
The prevalence of LSD in Malaysia, and its comparison with four other countries, is shown in Table 2. The prevalence of LSD among Malaysian was 0.43 per 100,000 live births or 1 in 231,904 live births. This rate was 26-fold lower compared to UAE (Asian) and Northern Portugal (European). Similar to this study, the most prevalent LSD in Northern Portugal, the Netherlands and the Czech Republic were sphingolipidoses and MPS. There were between-country differences in the prevalence of individual types of diagnosed LSD, not only between Malaysia, UAE and European countries, but also between UAE and the European countries. For example, Fabry and MPS II were the most prevalent in Malaysia; while GM 2 and Pompe were common in Northern Portugal and the Netherlands respectively. Among Asian population, GM 1 is ve times higher is UAE compared to Malaysian but MPS II is relatively high in Malaysia compared to UAE.
We found that Chinese were 17 times more likely to have Pompe disease compared to others (OR 16.98, 95% CI 3.52-81.88, p < 0.05). We noted out of 9 Pompe patients, 7 were Chinese and the c.1935C > A p.(Asp645Glu) was the pathogenic mutation detected in all of them.

Mutations Analysis
Sequencing analyses of the GAA, FUCA1, GBA, ARSA and GLNS genes had successfully identi ed mutations in LSD patients with low or undetected enzyme activity. Twenty-three (23) mutations associated with LSD were identi ed in Malaysia ( Table 3). The mutations detected include missense, nonsense, small deletions, duplications and splicing. In patients with MLD, three missense mutations, c,116dupG p.(Cys40Leufs*36), c.746 T > C p.(Phe249Ser) and c.922T > C p.(Tyr308His) were observed in ARSA gene which were also reported in patients with metachromatic leukodystrophy [12,13]. (Trp432*) in FUCA1 gene. Changes from T to A at nucleotide 393 was predicted to create a truncated α-L-fucosidase protein. This mutation was previously reported in two separate studies, involving patients of Chinese origin [14,15]. The c.1295G > A mutation caused substitution from amino acid Trp to a stop codon that subsequently led to a truncated protein. This mutation is located in a highly conserved region. Table 3 also shows distribution of the selected LSD mutations in Malaysian ethnicity. Pompe disorder was mainly presented in Chinese ethnicity. The three distinct mutations in GBA gene were found only in Malay ethnicity. As for MPS IVA, the mutation c.473_477delAGTGG and c.502G > T were found only in Malay ethnicity whereas mutation c.218A > G and c.551G > A were revealed in Indian ethnicity. Meanwhile, a few numbers of patients from Chinese descendant showed mutation c.106-111delCTGCTC and c.953T > G. Interestingly, fucosidosis mainly presented in three unrelated Iban patients and inherited from the parents involving homozygous p.(Trp432*) mutation in FUCA1gene.

Discussion
Our analysis suggested that the prevalence of LSD in Malaysia is quite low compared with the other four countries and the carrier frequencies is 1:241. We also observed that the most prevalent LSD subtype for MPS and sphingolipidoses in Malaysia were MPS II and Fabry, and the most prominent nucleotide change was Fucosidosis with c.1295G > A.
During the last 15 years, we are the only laboratory offering con rmational test for LSD in Malaysia. Our institution is currently the only centre with comprehensive LSD diagnostic tests comprises of screening and con rmatory tests in Malaysia. In addition, we also include two university hospitals (one in the Klang Valley and one in East Coast region), which provide the screening of MPS and send the presumptive patients' samples to us for con rmation. Majority of the most clinically suspected cases were from central region which mainly from Klang Valley. Therefore, a time tendency for a more uniform coverage of the country by our laboratory has been observed. Most clinically suspected cases from all part in Malaysia are referred to our centre for con rmatory diagnosis. Thus, our data highly re ective of the status of LSD in Malaysia. Furthermore, our data is the only representative data for LSD status in Malaysia as all clinically suspected cases from all part in Malaysia were referred to us.
The low prevalence of LSD maybe due to short period of data retrieval (2008-2017). The study of prevalence of LSD in The Netherlands [2], Czech Republic [16], United Arab Emirates (UAE) [17] and Northern Portugal [1], took more than 20 years. Clinical suspicion of LSD in patients among medical health practitioner especially general physician and paediatrician in Malaysia is still low and should be addressed by the respective authorities. Increase awareness of LSD and su cient laboratory facilities can help in detecting underdiagnosed patients. Geographical factor also plays some role as some patients from very remote areas may have di culties to access the medical facilities. This may had underestimated the prevalence of LSD in this region.
In Malaysia, mucopolysaccharidoses was the most common type of LSD (47 of 92 patients, or ~ 51%) with the prevalence of 1 in 292,401 live births. MPS Type II accounted the most among other MPS which showed similarity with China [18] and Taiwan [19]. Although sphingolipidoses were not common (0.13 per 100,000), prevalence of Fabry disease was almost similar to The Netherlands [2] and twice higher than UAE [17].
Over a 10-year period from 2008 to 2017, fucosidosis was the only disease detected (n = 4) in the oligosaccharidosis group. It was noted to be ~ 2-fold higher than in the Netherlands [2]. However, when comparing to UAE, the prevalence of fucosidosis in UAE was 20-fold higher than Malaysia [17]. This is most likely due to consanguinity that leads to a higher birth prevalence in autosomal recessive diseases with a presence of a 'founder mutation' in the ethnic group, as it is known that consanguineous marriage among Emiratis is common [17]. In this study, we observed that three out of four patients with fucosidosis originated from the state of Sarawak. The p.(Trp432*) mutation which caused amino acid substitution from Trp to stop codon in exon 8 of FUCA1 gene is predicted to cause a truncated protein [20]. We believe that this ndings may support our hypothesis of a founder gene p.(Trp432*) mutation among Sarawak population [21]. Holguin Province in Cuba has also been observed with highest incidence of fucosidosis with single mutation of p.(Gln422*) among its population [22].
Diagnosis of a few LSD appeared to be more delayed in Malaysia. For instance, the median age at diagnosis of MPS IVA and mucolipidoses type II in Malaysia were three times higher compared to Australia (9.3 years vs 2.7 years and 2.5 years vs 0.8 years respectively) [23]. Median age at diagnosis of MPS IIB (2.1 years vs 3.5 years) and Gaucher (4.5 years vs 9.5 years) were also higher than Australia. However, the range of age diagnosis in Malaysia was quite narrow compared to Australia (MPS IIIB: 1.17-3 years vs 0-21.4 years; Gaucher: 0.14-7 years vs 0-78.2 years) which maybe due to time of presentation of patient to the clinic. As for MPS I, MPS II, Pompe and MLD, the median age of diagnosis was almost similar between Malaysia and Australia. These results highlight that there is delay in the diagnosis of LSD in Malaysia.
We found that MLD was more common in the Iban ethnic population from Sarawak, while Pompe disease was more frequent in Chinese. The Iban population were 44 times more likely to have MLD compared to the non-Iban population (OR 43.53, 95% CI 15.29-123.93, p < 0.05). Therefore, the prevalence of MLD among the Iban population was estimated to be 1 in 6,981 or 14:100,000 live births. Few other studies have been conducted among ethnic populations around the world. Holve et al [24] reported that the incidence of MLD among Navajo tribes in United States was 1 in 2,520 live births, while the Habbanite Jews constituted a high-risk population for MLD with a reported incidence of 1 in 75 live births [25]. We were unable to calculate the prevalence of Pompe among the Chinese population due to inadequate data of live births among that population between 1988 to 2000.

Conclusions
These data are the rst to describe the prevalence, frequency and demographic data of patients diagnosed with LSD in Malaysia. In summary, LSD as a group, can be considered as not uncommon inborn error of metabolism in Malaysia with prominent certain types of LSD in certain ethnic; Pompe in Chinese community and MLD in Iban population. A more comprehensive study on the prevalence of LSD in Malaysia may provide valuable information.

Declarations
Ethical approval and consent to participate The need for approval was waived by the local ethical committee (Medical Review & Ethics Committee -MREC, Ministry of Health, Malaysia) as data were collected retrospectively and reported anonymously.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author after getting approval from the Director General of Health from Ministry of Health Malaysia.

Competing interest
All the authors declare that they have no con ict of interest disclose.   Total MPS  29  18  47  32  0  2  3  1  0  25  9