The Challenge of Diagnosis and Indication for Treatment in Fabry Disease

Introduction

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of alpha-galactosidase A (α-gal A) activity involved in the degradation of glycosphingolipids. Its deficiency leads to the accumulation of globotriaosylceramide (Gb3) inside the cells, causing several clinical abnormalities; the GLA gene is located on Xq22.1. ¹

The clinical suspicion of FD begins with a selection of characteristic signs and symptoms, such as acroparesthesia, angiokeratomas, recurring headache, abdominal pain, chronic diarrhea, progressive loss of renal function, cardiomyopathy generally associated with myocardial fibrosis, central nervous system microangiopathy, ^{1 –4} and/or positive family history, or also families with high prevalence of kidney disease, cardiomyopathy, or ischemic encephalopathy. ^1,2

After diagnostic hypothesis of FD is raised, the diagnosis is made using laboratory testing. In males, the enzymatic activity of α-gal A in plasma, leukocytes, skin biopsy, or dried blood spot (DBS) samples is measured. In females with clinical suspicion, it is widely described in literature that there is a limitation in performing enzymatic activity assay, since women can range from almost undetectable, as can be found in males, up to a normal range of values, as found in healthy individuals. ^{1,2,5 –7} In those patients, a diagnostic is performed with GLA gene molecular analysis.

Molecular analysis of the GLA gene is an essential diagnostic tool in women or in patients who have very strong clinical suspicion due to the presented symptoms but the enzyme activity is within the normal range. Furthermore, it can inform the treating physician about the exact kind of mutation, which is generally related to the clinical manifestations. Mutations known as “classic” have an earlier onset of symptoms, and usually cause a more severe clinical phenotype. On the other hand, “nonclassic” usually shows slower disease progression with later onset of symptoms but patients can also present severe organ damage. ⁸

The treating physician has to be aware of the different kinds of mutations and their clinical implications. Nonsense and frameshift mutations are supposed to cause a severe phenotype, since these mutations cause important damage to protein structure (misfolding), leading to a highly dysfunctional protein. ⁹ Mutations without previous description in literature require further investigation not only by performing brain and heart magnetic resonance imaging (MRI) but also by considering renal biopsy to evaluate which organs might be affected. There are also controversial mutations, like the p.D313Y and intronic variants, described in literature as pathogenic by some authors ¹⁰ and as benign by other authors. ¹¹ The treating physician should always rely on the patients’ clinical manifestations and examination, rather than the genotype, when deciding whether to begin enzyme replacement therapy (ERT). ¹²

Although there has been specific treatment over the last 15 years for FD, patients still face the challenge to have an early diagnosis that will allow a timely treatment initiation to avoid irreversible damage to organs. Treating physicians still have the challenge to decide what would be the ideal time to treat the later onset variants patients, and researchers of molecular alterations face the challenge of the unknown effect of GLA variants. We will try to address some of these challenges.

Biochemical Diagnosis

Determination of α-gal A activity is usually the first laboratory test for diagnosing FD in index patients; if there is a known familial GLA mutation, a straightforward DNA analysis is indicated. Many biological materials can be used for the analysis of α-gal A activity, but the most common are plasma, leukocytes, fibroblasts, and DBS. The assay in leukocytes is regarded as the “gold standard” and DBS assays are widely used for screening purposes.

As an X-linked disorder, male patients can be reliably diagnosed with FD if presenting low or undetectable α-gal A activity; results from DBS analysis below the normal range often require a confirmatory test on leukocytes and, if low α-gal A activity is also detected in this material, FD is confirmed. GLA sequencing for mutation identification is recommended for index cases, which could provide important insights for prognosis and treatment, as well as for genetic counseling but is not mandatory for diagnosis.

On the other hand, analysis of α-gal A activity in heterozygous females is usually not conclusive for Fabry diagnosis, as women may present normal enzyme levels in different samples due to random X-inactivation. Therefore, in those cases, identification of a pathogenic GLA mutation is mandatory to define the diagnosis and carrier status of a female patient. Additional complementary tests such as measuring Gb3 and its metabolites (mainly lyso-Gb3) in urine/plasma and biopsies for histology/electron microscopy are also recommended. ¹³

Lyso-Gb3 is similar to Gb3, only lacking the fatty acid chain. ¹⁴ Levels of plasma lyso-Gb3 have been correlated with disease severity as well as with response to ERT. ^15,16 In male patients, lyso-Gb3 is a reliable diagnostic tool to distinguish between classical and nonclassical FD from non-FD participants. However, in some cases of female patients, the levels of lyso-Gb3 overlap with controls, suggesting that increased lyso-Gb3 values are very suggestive of FD, but normal values cannot exclude FD. ¹⁷ Urinary lyso-Gb3 levels and its analogs are lower than plasma, but it has been demonstrated that male patients present higher levels of these markers when compared to females, and none of these markers are found in healthy individuals. ¹⁸ In addition, increases in lyso-Gb3/creatinine ratio correlates with Gb3 concentration, types of GLA mutation, gender, and ERT status, ¹⁹ emphasizing the diagnostic value of this biomarker.

Molecular Diagnosis

The human GLA gene is organized into 7 exons encompassing over 12 kb; the exons range in length from 92 to 291 bp. There are several possible regulatory elements in the 5′-flanking region such as, “CCAAT” box, enhancer elements, and sequences corresponding to the activator protein 1, octanucleotide, and “core” enhancer element but no typical “TATA” box. Moreover, the GLA gene has an unmethylated CpG-rich island upstream of the initiation codon. ³⁷ The messenger RNA encodes a protein of 429 amino acids, including the N-terminal signal peptide of 31 amino acids. ^{37 –39} After cleavage of the signal peptide, posttranslational modifications occur in the Golgi and lysosomes and a mature protein is formed. ⁴⁰

Mutations in the GLA gene have been identified using DNA sequencing. The 7 exons of GLA gene are amplified by polymerase chain reaction. Sense and antisense oligonucleotide primers are synthesized based on the sequences flanking the 7 exons of the GLA gene. Sequences are compared with the reference sequence NG_007119 (http://www.ncbi.nih.gov) and confirmed by reverse strand sequencing. To date, more than 840 mutations have been reported in the GLA gene in the Human Gene Mutation Database, ⁴¹ that are believed to cause FD. Of these, 589 are missense and nonsense mutations, 42 are splicing substitutions, 4 are regulatory substitutions, 114 are small deletions, 40 are small insertions, 11 are small indels, 35 are gross indels, 4 are gross insertions, and 6 are complex rearrangements.

In a study conducted in our laboratory with 568 individuals from 102 families with suspected FD, we found 51 families presenting 38 different alterations in the GLA gene, among which 19 were not previously reported in the literature. ⁴² Most mutations are detected in only 1 family, and it is necessary to determine the physiological impact of these new mutations. In order to predict the possible impact of the new mutations on the structure and function of a human protein, different silico predictors are used.

In addition, in our sample we found an intronic haplotype with 4 variants described in individuals with suspected FD. One variant, the c.-10C>T (rs2071225), was found in the untranslated region of exon 1. It is described in the literature as polymorphism and appears in 12% of the population. ⁴³ (The single nucleotide polymorphism database [dbSNP] can be found at http://www.ncbi.nlm.nih.gov/SNP.) The variants c.370-77_370-81delCAGCC (rs5903184), c.640-16A>G (rs2071397), and c.1000-22C>T (rs2071228) are also found in intronic and are described in the literature as nonpathogenic polymorphisms (dbSNP). Although these variants are described as nonpathogenic, the impact of these changes together is unknown. Considering that these regions are not routinely evaluated by gene sequencing, the prevalence of FD may be underestimated. ⁴⁴

Indications for Treatment

Many questions about the optimal time to initiate FD treatment are still present in clinical practice. The main studies guide the physician who has the autonomy of therapeutic decision as he or she often already knows the family history and the complications involved.

Information about the evolution of the disease is not always available, so some suggestions can be applied. First, the differentiation according to gender should be made by the team that treats a patient with FD. Another feature to be considered is whether the disease presents itself in classical or nonclassical form, as described above. In most consensus and expert opinions on FD, those with classical or nonclassical disease should be treated as soon as the first target organ impairment signals are noted, such as in the kidney, heart, or central nervous system. ^{6,53 –55} In Brazil, for all ages greater than or equal to 18, treatment should be indicated if the following are present:

In relation to a glomerular filtration rate indicating hyperfiltration was excluded as an essential criterion for treatment of FD because it is an unspecific and unclear clinical relevance finding, even considered a confounding factor for some nephrologists. ⁵⁵

Another topic to be discussed is the indication of treatment for children. An ERT is safe for this age-group. ⁵⁹ The initiation of treatment among this group is also controversial. Studies show a lower accumulation of kidney substrate, especially if ERT starts before the age of 10. Recent research has shown that early treatment slows disease progression. ^{57,60 –62}

When the diagnosis is made by neonatal or family screening or its mutation is not a classic one, medical decisions should be based on clinical information of other family members if available. However, an intravenous treatment can be initiated early, before symptoms can decrease school performance and lead children to being labeled chronically ill. ⁶³ Thus, the decision to treat should be careful, involving the patient, family, and health-care team. ⁵⁹ A good doctor–patient relationship is the key to success and the decision to start ERT must be evaluated on a case-by-case basis by physicians. ⁵⁴

The treatment can be considered mainly for boys between 8 and 10 years old with classical FD. There are renal deposit findings before microalbuminuria begins. ^59,64 In children with nonclassic disease, the current recommendation is frequent monitoring with different medical specialties. That treatment should be promptly initiated when symptoms emerge or in the presence of an abnormal renal biopsy. ⁵⁹

Conclusion

The challenges in the diagnosis and indications for treatment of FD are part of today’s clinical practice. Thus far, we have learned that the enzymatic assay is diagnostic for men, while women most often need the molecular study to have a definite diagnosis. The enzyme activity assay in DBS has its established use in case studies in high-risk populations due to the stability of the enzyme in this kind of sample and ease of shipping. The effects of controversial mutations in exons or multiple intronic are not yet fully understood and seem to have individual variations. In this situation, the disease staging of the patient will give us the answer if the ERT is indicated or not. We believe today that treatment is indicated in children with acroparesthesia due to the relationship that seems to exist with their presence and renal impairment. In conclusion, FD is still being studied and in the medium-/long-term, we will have some answers and probably more questions.