A New Genetic Insight for Orphan Renal Disorder, Fabry: A Review

Fabry is the rare X-linked genetic disorder caused due to mutation in Alpha –Galactosidase encoding GLA gene mutation in chromosome number 22. It has wide diversification in prevalence due to clinical heterozygosity. There are some potential biomarkers for the evaluation of normal or altered genes responsible for Fabry. Advances in the research of biomarkers over the years have made significant development for several clinical indicators, viz. urine-derived cells, oxidative stress, DNA methylation, etc. At present days the recommended therapies for the disease are Enzyme Replacement Therapy (ERT), Chaperone therapy (CT), and mRNA-based therapy, besides, some second-generation therapies which are still under clinical trials.


INTRODUCTION
Fabry disease is a rare renal disorder caused by mutation of the X-linked GLA gene which results in the deficiency of alpha-galactosidase [1]. The cells which are deficient in the alphagalactosidase enzyme can not metabolize glycosphingolipid mainly Globotriaosylceramide (GB 3 ) [2][3][4][5][6][7]. Accumulation of GB 3 can lead to various symptoms and for the progression of the cascade (series of progressive events) rather than deficiency of enzymes so maybe a diagnostic tool for the detection of Fabry disease [8]. GB 3 cannot gain its original metabolized form Alpha-D-Galactosyl residue which is the end product of the reaction and gets clustered in

Review Article
higher accumulation of Globotriaosylceramide in lysosomes of cells and tissues. It belongs to renal lipidosis and may accumulate into different organs which causes multisystem disease [9].
Fabry prevalence is associated with a wide range in white male population approximately 1:17000 to 1:117000, in which early manifestation mutation is seen in 1:22000 to 1:40,000 in males; advance onset manifestation has been reported in about 1:6000 to 1:40,000 in females and 1:1000 to 1:3000 in males [9]. European database which reports about check on the efficiency, potential, and safety of Enzyme Replacement Therapy with algalsidase alpha and considering to monitor cardiac events in Anderson Fabry Disease is an outcome of "Fabry Outcome Survey" [10].
In India, the estimated data for the prevalence of Fabry disease is 1/40000 to 1/117000 is of live birth and includes both classical and nonclassical phenotypes [11].
There are two major forms of Fabry based on enzymatic activity-
The rarity of Fabry made it difficult to understand the mechanism. Age and onset are variable, a person with unknown family history is diagnosed in later life, the reason for later onset detection is the slower progression of detectable alphagalactosidase and causes less damage to the organs. Glomerular mesangial illness, vacuolated epithelial cells, lipid inclusions are the signs of renal abnormality. The early detection of proteinuria is the apparent sign of abnormal renal dysfunction that leads to renal failure in many individuals before the treatment methodology was developed like Enzyme replacement therapy [12].

GENETIC ARCHITECTURE OF FABRY
There may be huge clinical variations among the same family mutation which creates difficulty in its diagnosis and men can be diagnosed only if the residual enzyme activity is found to be <35% of the mean. Females' heterozygote shows high enzyme activity due to mosaic (random inactivation) in later stage and which shows the involvement of few organs only [14]. Gla variant with unclear significance is also present and termed as a fringe allele [15].
The deficiency of alpha-galactosidase (GLA) lysosomal hydrolase enzyme (EC 3.2.1.22) causes the accumulation of GB 3 (alpha-linked Galactosyl moiety). 3 Prenatal diagnosis and heterozygous carrier identification, candidate identification for enzyme replacement therapy can be made easier with the help of mutation heterogeneity provides molecular pedigree analysis and stability properties [16]. The period involved from the visibility of the first symptom to its proper diagnosis is approximately 15 years, sometimes it may vary from individual to individual and severity of the disease [13]. Alpha-galactosidase enzyme has approx. 1318 nucleotide in the X-Chromosome q22.1 which encodes 429 amino acid long protein chain. In this sequence, 1-31 act as signal peptides, and 32-429 form the main protein. An amino acid from 203 to 207 makes substrate binding region, amino acid 139, 192 & 215 are glycosylation region and amino acid from 170 to 231 makes active region. This region is very important for the normal function of protein and mutation in the region as shown in Table 3, and

Aetiology of GLA Gene and Genomic Variants
A large variety of polymorphisms ( Fig. 2 and Table 3) have been reported in GLA variants. Some intronic mutations have been reported as a pathogenic variant of the GLA gene and the mode of mutation comprises nonsense, splice mutation, rearrangements, deletion, and duplication in a wider range [19].
Asymptomatic: 1208 delta 3, is the frameshift mutation due to deletion of arginine 404 in a polypeptide of the residual enzyme [10].

Novel Genomic Variants of Fabry Disease
The c.448delG is the de novo mutation, found as a result of a deletion in exon 3 of the GLA gene. Some of the symptoms include vertigo, acroparesthesia, cornea verticilata, and abdominal main [20].

1.
In the population of Argentina, two nonclassical missense mutations were foundp.(Cys174Gly): This mutation causes unfolding by disturbing thermal stability and as a result protein aggregates lead to organ damage [21].

p.(Arg363His):
In this case heat shock protein (Hsp 70) in normal condition binds to Arginine (positively charged amino acid) but in mutant form Histidine 363 is unable to bind properly with Hsp 70 causes instability in protein and the transportation to lysosome does not take place [21].

Alpha GAL A:
Intron splicing mutational c.801 C 1G > A-It is an insertion mutation that causes shifting of the frame by addition of 36 nucleotide termination sequence TGA in the Exon 5 placing 12 nucleotides downstream causes formation of 270 amino acid residue of truncated GLA protein that declines the mRNA stability with the nonsense-mediated decay approach [22].

Genomic variants in India:
The genotypic analysis of 54 patients from 37 families were done in India and the data was taken from 10 territory referral centers which revealed 33

CLINICAL FEATURE
Fabry manifestation has been reported both in males and females; males with severe renal dysfunction and female with heterozygote in early-stage shows renal insufficiency and random inactivation in advance stage besides, non-classical symptoms involving target organs [14].

Threshold
The appropriate threshold for the detection of Fabry considers 30-35% alpha-galactosidase enzyme activity as the cut-off for diagnosis and for frequent measurement of enzyme activity dry blood spots or peripheral leucocytes are suitable samples [14,24].
The major value for pathogenicity in the form of threshold is still unknown as it varies depending upon the individual to individual and also upon the organ involved [24].

Associated Risk
Individuals' risk for complications depends not only on the alpha-galactosidase but also on other factors like epigenetic, environmental, and genetic factors which are considered to be the risk factors for the prevalence of Fabry disease [14].
The MRI (Magnetic Resonance Image) of the brain shows the presence of an ischemic cerebral lesion, due to several mutations detected by genetic polymorphism (Table 4) [25,26].
Factor V Leiden mutation and alphagalactosidase a activity which was seen subsequently in the mouse model confirmed the enhanced thromboembolism along with up-regulation of vascular thrombi level which was not seen in the mouse model alone [14].

Symptoms
Renal disease is considered to be a potent convict for mortality and morbidity among individuals with Fabry disease [27]. Missense mutation GLA:c657C>G:pIle219Met 2.

Fig. 2. GLA: Gene to prot
Gene Image were captured from NCBI ( the protein sequence from uniprot (p06280) As per the diagnosis cascade and screening of relatives for phenocopy identification in patients with inherited cardiomyopathies regardless of the age of the patient, it becomes evident that chromosome inactivation mostly did not manifest any effect in the female carrier but due to random inactivation some discrete symptoms have been observed in affected females [28]. Renal disease is considered to be a potent cause for mortality and morbidity among individuals suffering from Fabry disease [27]. There is enormous variability within clinical courses and symptoms (Fig. 3) of affected people suffering from Fabry disease and the basis of morbidity could be determined with As per the diagnosis cascade and screening of relatives for phenocopy identification in patients with inherited cardiomyopathies regardless of the age of the patient, it becomes evident that chromosome inactivation mostly did not manifest any effect in the female carrier but due to random some discrete symptoms have been ]. Renal disease is considered to be a potent cause for mortality and morbidity among individuals suffering from ]. There is enormous variability symptoms (Fig. 3) of affected people suffering from Fabry disease and the basis of morbidity could be determined with the help of positive family history, severity which leads to morbidity condition showing involvement of renal, cerebrovascular and cardia

GLA: Gene to protein sequence and its variations
The affected male shows symptoms like proteinuria, microhematuria, and lipiduria, angiokeratomas, edema, abdominal pain, and acroparesthesia is more often seen in childhood. The main factor for elevated risk is premature stroke, transient ischemic attack, and dementia [29].
Pathogenicity like myocardial, neurophysiological, cerebrovascular, classical like many symptoms have been observed in the FD patients are useful for diagnosis. The affected male shows symptoms like proteinuria, microhematuria, and lipiduria, angiokeratomas, edema, abdominal pain, and acroparesthesia is more often seen in childhood. The main factor for elevated risk is premature hemic attack, and dementia Pathogenicity like myocardial, neurophysiological, cerebrovascular, classicallike many symptoms have been observed in the FD patients are useful for diagnosis.

GB 3 Accumulation
To check or monitor the efficacy of therapy and the progression of the disease is being monitored with the help of measurement of GB3 accululaion [30].
GB3 accumulation is the potent biomarker because of its direct use as it releases secondary mediators for glomerular Fabry nephropathy and causes disease pathogenesis and progression through fibrosis and inflammation stimulation [30]. In vitro analysis of prime urine-derived cells showed elevation in the expression level of COL4, FN1, HES1, and TGFβ1 [31].

Primary Urine-Derived Cells
Urine cells are non-invasive cells of a patient with Fabry disease decreases Alphagalactosidase enzyme activity and that mimics the cellular in vivo model and dysregulation of lysosomal protein, so the finding is based on proteomics, qRT-PCR, western blot, immunoassay, and this may be the prospects for the diagnostic purpose as both show Globotriaosylceramide accumulation (lysosomal storage material) [32].

Oxidative Stress Biomarkers
Several mutations are found related to oxidative stress revealed the involvement of smooth muscle cells, cardiac myocytes, and all types of renal cells such as podocytes, tubular and glomerular cells. Oxidative stress is responsible for organ damage is a hypothesis considered by several authors [33].
Occurrence of pro-oxidant state induced (Table-5) by Lysosomal Gb3 in Fabry patient had been demonstrated by Biancini [34]. A positive correlation was found as the level of advanced oxidation product increases the reduction of thiol group and ferric reducing power [33], results in a reduction of proteins responsible for oxidative damage [34]. The involvement of mitochondrial DNA haplogroups for modulation of oxidative stress in patients suffering from Fabry was established before the Enzyme replacement event [35]. Elevated Lipid peroxidation showed irreversible effect after Enzyme replacement therapy as per hypothesis [36].
Cardiovascular renal remodeling pathophysiology suggested oxidation stress signaling cascade and activation of oxidative stress and p22phox expression were reported substantially elevated by Ravarotto, et al. [37] Table 6 shows the diagnostic approaches and Table 7 shows the emerging potential tools in the study for diagnosis purposes.  Increase in GFR-Early detection tool [38]. A decline in GFR-Later stage [39]. Two different strategies based on serum creatinine equation:-Adult-Chronic Kidney Disease Epidemiology Collaboration(CKD-EPI) [40].

3.
Cystatin -C Cystatin-cis the potent inhibitor act as a protease inhibitor found in all nucleated cells and is useful for the diagnosis of early-onset that is classical renal dysfunction diagnosis. It can decrease the GFR to a little extent in both males and females [43].

4.
Urine microscopy Phase-contrast microscope-Tubular epithelial cells were seen. Polarized microscope -Urine sediments observation showed Maltese cross bodies encapsulated in the mulberry cells [42].

5.
Renal biopsy Renal biopsy(histological study)is the most trustable tool. Stereomicroscopy-For more sensitive histological renal structures stereomicroscope is used. Electron Microscopy-Zebra bodies (small osmophilic structures) were observed which were seen encased in a coated membrane [42].

6.
Proteinuria Amiloride has an important function in vitro to reduce podocyte (renal cells) motility and it causes proteinuria in mice [43]. Epigenomics Fabry patient's nephropathy from urine sample determination by comparing with control revealed the presence of a greater number of post-translational modifications such as GB3 methylated isoforms found in urine and plasma of the patient [46].
The study of proteomics is quite easy in patients with Fabry disease because of its noninvasiveness, and no limit on the number of samples to be used [48].

4.
Metabolomics Male individuals have a higher amount of metabolites in urine samples as gender influences Urine level in pediatric patients [49].

Potential Biomarker
 Bikunin-Bikunin is the Urine Protease inhibitor(serine protease).Bikunin was found elevated in the Fabrypatients compared to the individuals with other renal impairment. The elevated bikunin along with proteinuria is used as a novel biomarker [50] (Fig. 4).  Urine Podocyte-As the glomerular impairment progresses in Fabry patients, the podocytes enter into urine and resulting in an elevated concentration of urine podocytes. The latter condition is known as podocytinuria (Fig. 4) [  Pelvic cyst-Renal impairment correlates with the pelvic cyst reported by regular monitoring with ultrasonography. It is not the only pathognomic or significant tool for Fabry diagnosis but their presence can recruit further diagnosis for Fabry (Fig  [3].  Tubular Protein-Type IV collagen and Transferrin protein leakage has been reported in correlation with GFR and their presence in urine may be considered as the novelty for the diagnostic approach (Fig. 4) [4].

Fig. 4. Potential marker for diagnosis
Bikunin is the Urine Protease inhibitor(serine protease).Bikunin was found elevated in the Fabrypatients compared to the individuals with other renal impairment. The elevated bikunin along with proteinuria is used as a novel As the glomerular impairment progresses in Fabry patients, the podocytes enter into urine and resulting in an elevated concentration of urine podocytes. The latter condition is . 4) [2]. ent correlates with the pelvic cyst reported by regular monitoring with ultrasonography. It is not the only pathognomic or significant tool for Fabry diagnosis but their presence can recruit further diagnosis for Fabry (Fig. 4) V collagen and protein leakage has been reported in correlation with GFR and their presence in urine may be considered as the novelty for the diagnostic approach  Glomerular Protein-Microglobulin and N Glucosaminidase and Alanine aminopeptidase are the glomerular proteins and their dysfunction can be indicated as the sign of Fabry renal impairment (Fig. 4) [5].  Differential DNA Methylation DNA Methylation is also a potential characteristic for rare diseases to detect renal anagenesis, glomerular disease, proteinuria, and membranous nephropathy (Fig. 4) [6].  6. TREATMENT

Systemic mRNA (Messenger RNA) Therapy
From the preclinical studies, it was revealed that intravenous administration of encoding systemic mRNA helps to promote the production of the alpha-galactosidase enzyme in mice and humans. It was found thermostable and capable of substrate reduction (accumulated GB3 reduction) as an alpha enzyme in Fabry patients who lack wild alpha-galactosidase enzyme. This approach is

Systemic mRNA (Messenger RNA)
From the preclinical studies, it was revealed that intravenous administration of human GLA encoding systemic mRNA helps to promote the galactosidase enzyme in mice and humans. It was found thermostable and capable of substrate reduction (accumulated GB3 reduction) as an alpha-galactosidase ts who lack wild-type galactosidase enzyme. This approach is also advisable for use in other lysosomal storage disorders [51].

Enzyme Replacement Therapy
Enzyme replacement therapy (ERT) has been in practice since 2001, which implies the introduction of recombinant GLA into the host (Fabry patient) once in two weeks intravenously. It can revert the abnormalities a dysfunctionalities [52]. ERT is a costly procedure and requires a lot of skill, most effective in the treatment of people with cardiac and renal Fabry disease at classical onset, however, its non classical/later-onset effect is not known [ Regular administration is required as these enzyme does not have a significant half The two major enzymes are used replacement therapy-

Chaperone Therapy
Frustaci et al. performed the first clinical trial as pharmacological chaperon therapy using galactose [55]. In Europe and Canada the

Replacement Therapy
(ERT) has been in 2001, which implies the introduction of recombinant GLA into the host (Fabry patient) once in two weeks intravenously. It can revert the abnormalities and metabolic ERT is a costly procedure and requires a lot of skill, most effective in the people with cardiac and renal Fabry disease at classical onset, however, its nononset effect is not known [52]. Regular administration is required as these t have a significant half-life [53].
The two major enzymes are used for enzyme alfa "Replagal" (Shire Human Genetic Therapies, Cambridge, A) -0.2 mg/kg administration in each infusion.
beta "Fabrazyme", (Genzyme Corporation, Cambridge, MA) -1 mg/kg each infusion [54] (Food and drug administration the ly the use of Fabrazyme . performed the first clinical trial as pharmacological chaperon therapy using [55]. In Europe and Canada the approval was given to the pharmacological migalastat (Galafold™; Amicus Therapeutics, Cranbury, NJ, USA) for treatment of Fabry disease caused due to amenable mutation [56].
During the study of misfolding mutation in the enzyme, to enhance the intracellular activity of mutant enzyme the competitive inhibitor application (results in the proper folding of mutant enzyme, protein maturation as well as transportation to the lysosomal) approach is applied to treat Fabry disease st misfolding mutant enzyme [57].

1-Deoxygalactonojirimycin
(DGJ) is the competitive inhibitor of the potent alpha galactosidase enzyme that binds to the active site of the enzyme and causes its proper folding as well as intracellular targeting to the lysosome for the degradation of accumulated GB3 in mammals. It has been proved during the study of transgenic mice with the similar galactosidase enzyme (misfolding mutant) by oral administration [58]. Seventy-eight misfolding mutations have been treated with this approach, some of them are assayed with patient's cells A20P, N34S, R49L, F113L, Y207C [59] andR49C, M51K, I91T [60].

Substrate Reduction Therapy
SRT maintenance therapy requires less dependency on enzyme infusion consequently, it shows excellence in improvement in the quality of life as it circumvents ERT (Enzyme replacement therapy) [61].

Methodologies of fabry treatment
; Article no. JPRI.78861 approval was given to the pharmacological (Galafold™; Amicus Therapeutics, Cranbury, NJ, USA) for treatment of Fabry disease caused due to amenable mutation [56].
During the study of misfolding mutation in the zyme, to enhance the intracellular activity of mutant enzyme the competitive inhibitor application (results in the proper folding of mutant enzyme, protein maturation as well as transportation to the lysosomal) approach is applied to treat Fabry disease strictly with Deoxygalactonojirimycin (DGJ) is the competitive inhibitor of the potent alphaenzyme that binds to the active site of the enzyme and causes its proper folding as well as intracellular targeting to the lysosome for the degradation of accumulated GB3 in mammals. It has been proved during the study of transgenic mice with the similar alphagalactosidase enzyme (misfolding mutant) by eight misfolding mutations have been treated with this approach, some of them are assayed with patient's cells -A20P, N34S, R49L, F113L, Y207C [59]

Substrate Reduction Therapy
SRT maintenance therapy requires less dependency on enzyme infusion consequently, it shows excellence in improvement in the quality of life as it circumvents ERT (Enzyme In-vivo study in a mouse model with early neonatal death indistinguishably showed GB3 accumulation in kidney and vasculature due to lack of an alpha-galactosidase enzyme in Fabry patients. Lymphoblasts were transformed from Fabry disease patients with EtDO-P4 showed a significant reduction of glucocerebroside and globotriaosylceramide.

Second-Generation Therapy
Pegunigalsidase-alfa: Cells of tobacco plant produces Pegunigalsidase-alfa (PRX-102) which was modified as fusogen (PEG) polyethylene glycol for maintenance of stability and to tolerate reduction from clearance was confirmed by experiment on Human plasma. The murine model showed an extra reduction in the GB3 level as compared to an alpha-galactosidase enzyme with the help of PRX 102 in plasma of Fabry patients [62].
Moss-aGal: R-αGAL's, a plant-derived form is a moss a Gal having immense potential of enzyme activity, and it has the property to be absorbed by endothelial cells. Moss aGAL has higher enzymatic activity than alphagalactosidase as revealed from the studies performed on the FD mice model [63] and is much more effective for clearing accumulated GB3 than alpha-galactosidase. In a single infusion, moss aGAL has a half-life of 14 minutes in plasma as detected by pharmacokinetics, less than normal alpha-galactosidase enzyme [61] (Fig. 5).

CONCLUSION
Clinical heterogeneity of Fabry Disease endorses the individualized diagnostic approach discovery with transcriptomics, epigenomics, metabolomics based on the patient's age, gender, type of mutation, and the onset of manifestation. The diagnostic tools utilize oxidative stress biomarkers, elevated GB3, some novel biomarkers like bikunin, tubular and glomerular cellular concentration, urine podocytes, cystatin C, and hold potential promise for Fabry diagnosis and will further expand in foreseeable with several mutations in GLA gene. The classical ERT(Enzyme Replacement Therapy), Chaperone therapy and recently mRNA-based therapy, substrate reduction therapy, secondgeneration therapy like Pegunigalsidase-alfa, Moss-aGal have the most significant clinical benefits. The conundrums of rarity summarize the need for progressive research and development.

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