Endomyocardial biopsies in patients with left ventricular hypertrophy and a common Chinese later-onset fabry mutation (IVS4 + 919G > A)

Fabry disease (MIM 301500) is an X-linked lysosomal storage disorder resulting from deficient alpha-galactosidase A (α-Gal A) activity. The estimated incidence of classic Fabry disease is 1 in 40,000-60,000 males in the general population [1]. The deficient α-Gal A activity results in progressive accumulation of glycosphingolipid, predominantly globotriaosylceramide (Gb3), in the walls of small blood vessels, nerves, dorsal root ganglia, renal glomerular and tubular epithelial cells, and cardiomyocytes. Clinical features in classically affected patients include acroparesthesia, angiokeratoma, and hypohidrosis in early childhood or adolescence and progress to renal insufficiency, cardiomyopathy, and cerebrovascular disease in adulthood [2‐6]. Recently, several different later-onset phenotypes of Fabry disease have been identified, which have drawn the attention of more physicians [7‐9]. Patients with later-onset Fabry disease have higher residual enzyme activities than those of the classical type. They lack the classic symptoms of Fabry disease and present relatively fewer or isolated symptoms such as hypertrophic cardiomyopathy, renal failure, or cryptogenic stroke at later stages in life [10‐14]. The cardiac later-onset phenotype usually presents only with cardiac manifestations, such as hypertrophic cardiomyopathy, mitral insufficiency and/or arrhythmias in the fifth to eighth decade [12, 15, 16].

In Taiwan, our team first revealed a surprisingly high incidence (approximately 1 in 1600 males) of a later-onset GLA mutation, IVS4 + 919G > A (GenBank accession nos. CS020811), in our population [17] and also identified this mutation in a number of Taiwan Chinese adult patients with idiopathic hypertrophic cardiomyopathy [18]. Thereafter, another newborn screening center of Taiwan also revealed a very similar incidence (1 in 1,460 males) of this mutation in their study [19]. More recently, DNA-based newborn screening for this mutation revealed a higher incidence (1/875 in males and 1/399 in females) than our previous enzyme-based Fabry newborn screening in Taiwan [20]. Because of the high prevalence of this IVS4 + 919G > A mutation in our population [21, 22], we analyzed the disease onset rate according to the ages of the male and female adults with this mutation. 86 male and 248 female adults who were older than 30 years were enrolled in this analysis. We found the disease onset rate for both male and female patients increased gradually with increasing age (Figure 1). Around 77% of male adults and 35% of female adults, who were older than 40 years old, had already developed hypertrophic cardiomyopathy. In particular, 94%(32/34) of the males who were older than 60 years old and 100% (6/6) who were older than 70 years old showed significant hypertrophic cardiomyopathy (Figure 1). We believe this result strongly suggests that this IVS4 + 919G > A is a real pathogenic mutation. Although, the IVS4 + 919G > A mutation was found in such a high frequency, the pathologic changes in later-onset Fabry disease with this specific mutation is still poorly known. It is important to look into the pathologic characteristics and changes of cardiomyopathy in these patients [23].

Due to the high prevalence of IVS4 + 919G > A mutation in our population, a new treatment guideline has been set up by the Bureau of National Health Insurance in Taiwan. All Fabry patients applying for enzyme replacement therapy (ERT) funding from the national health insurance have to receive an endomyocardial biopsy examination to demonstrate that Fabry disease is the primary cause of their hypertrophic cardiomyopathy. This guideline gives us the opportunity to observe a large series of cardiac pathologic changes in these patients with left ventricular hypertrophy and the IVS4 + 919G > A mutation.

The demographic data and clinical manifestations of these patients are shown in Table 1. Interestingly, high prevalences of hypertension and hyperlipidemia were noted in these IVS4 + 919G > A patients with hypertrophic cardiomyopathy. Out of these 22 patients, 12 (54.5%) were noted to have hypertension. All of these hypertensive patients received anti-hypertensive treatment and were kept normotensive (<140/90 mmHg) for at least 6 months before commencement of ERT. Seven (31.8%) patients were found to have hyperlipidemia and all of them were well controlled by medication for at least 6 months.

Twenty out of 22 patients received gadolinium-contrast enhancement cardiac MRI with LE technique and 6 patients (30.0%) were found to have severe fibrosis which is defined as at least 2 segments that are positive in myocardium delayed enhancement scan.All the endomyocardial biopsies were successfully performed without complication. For the pathologic change of the endomyocardial biopsies, all 22 patients (100%) showed myocardial hypertrophic change with distorted nuclei, and increased cardiomyocyte size with H&E staining. Five (100%) of the patients who did not receive ERT showed diffuse vacuolization in their myocardiocytes (Figure 2A) which is a common pathologic finding for Gb3 accumulation in cardiac biopsies of Fabry patients. In the other 17 patients who had received ERT, except for 3 patients (nos. 16, 17 and 22) who had received ERT for more than 3 years (Figure 3), all of them also showed diffuse Gb3 accumulation in cardiomyocytes. Regarding the toluidine blue staining, except those 3 patients (nos. 16, 17 and 22), the other 19 patients were also noted to have a lot of dark blue substance accumulation in cardiomyocytes, which is consistent with Gb3 accumulation (Figure 2C and D). In addition to Gb3 accumulation, patient no. 10 was found to have interstitial fibrosis among the cardiomyocytes with H&E staining (Figure 2B). Regarding the electron microscopic examination, except for those three patients, all other patients were found to have abundant lamellar myelin bodies in their cardiomyocytes (Figure 4). Furthermore, 14 patients (63.6%) were found to have significant myofibrillolysis (Figure 4D). There was no Gb3 accumulation in the capillary endothelium of any of these patients (Figure 4E, and F), which is compatible with the histopathologic finding of later-onset Fabry disease.

The diameters of cardiomyocytes were measured in all 22 patients (Figure 5). The average diameter of the cardiomyocytes was 25.35 ± 6.02 μm. The result of each individual is shown in Table 1. The average diameter of the control cardiomyocytes was 14.89 ± 2.63 μm.

Recently, the later-onset phenotypes of Fabry disease were found to be about 10 times more frequent in most populations [1, 20, 42, 43]. Owing to the characteristics, the terminology “later-onset” Fabry disease might be more appropriate than “variant” Fabry disease. In fact, an increasing number of novel mutations have been found and believed to be later-onset mutations through high-risk patient screenings or newborn screenings. On the other hand, some mutations, which were previously thought to be later-onset mutations, have been proved to be non-pathogenic. The most noticeable example is the p,E66Q mutation (c.196G > C), a previously identified renal later onset type mutation, which has a very high prevalence rate in Japanese and Korean populations (0.5-1%) and which has now been proved by pathologic studies to be merely a single nucleotide polymorphism, not a pathogenic mutation [44]. Therefore, it is insufficient to confirm a later onset mutation based on its enzyme activity and clinical manifestations such as hypertrophic cardiomyopathy or renal impairment.

Biomarkers, such as Gb3 or lyso-Gb3 were considered to be helpful to determine if the novel mutation is a true pathogenic mutation [27]. However, several reports indicated that Gb3, and even lysoGb3 might be normal in certain Fabry patients who had developed significant clinical manifestations with the pathogenic mutations [45, 46]. Renal or cardiac biopsy can provide more information to identify if a novel mutation is a real pathogenic mutation. In the retrospective single-center direct observational study, we reviewed the endomyocardial biopsies of large numbers of later-onset Fabry patients with IVS4 + 919G > A mutation to prove this mutation is a real pathogenic mutation.

Cardiac biopsy of classical Fabry disease patients revealed significant Gb3 accumulation in both endothelium and cardiomyocytes [47‐53]. Furthermore, the degree of endothelial Gb3 deposition was even used as a scoring system for monitoring the long-term cardiac therapeutic outcomes of ERT in classic Fabry disease (Thurberg, B.L. et al) [54]. In contrast to classical Fabry disease, cardiac biopsy of the later-onset patients revealed that Gb3 accumulation was confined to the myocardium and did not involve endothelial cells [55]. Other reports have also showed no endothelial Gb3 accumulation in later-onset Fabry disease [56‐58]. Therefore, no endothelial Gb3 deposition is expected in later-onset Fabry patients. The cardiac biopsies of our patients showed significant Gb3 accumulation in their cardiomyocytes but not in endothelial cells. It could indicate that IVS4 + 919G > A mutation is a true later-onset Fabry mutation.

Interestingly, a high frequency of hypertension (12 of 22; 54%) and hyperlipidemia (7 of 22; 32%) were noted among these cardiomyopathic patients with the IVS4 + 919G > A mutation. In this study, we did not check other genetic causes of hypertrophic cardiomyopathy. Actually, the exact causal relationship of these factors with hypertrophic cardiomyopathy in our patients has not established. Because most of our patients did not have significant renal impairment, the hypertension was not likely caused by Fabry renopathy. Several studies have indicated that in addition to renal impairment, hypertension could be caused by the imbalance of the autonomic nervous system in patients with Fabry disease [59]. Further studies are required to clarify the relationship between hypertension and the autonomic nervous imbalance in these patients. Furthermore, hypertension might be a predisposing factor to the development of hypertrophic cardiomyopathy for these later-onset Fabry disease patients, especially for the milder Fabry patients. The finding that four out of our five (80%) female patients had significant hypertension supports this finding. In addition, several IVS4 + 919G > A patients in our studies were found to be hypertensive. However, due to the small sample size in our study, we cannot make a definite conclusion regarding the relationship between hypertrophic cardiomyopathy and hypertension, more data will need to be collected and analyzed to clarify this point. Regarding predisposing factors for hypertrophic cardiomyopathy in later-onset Fabry patients, a recent important study, reported by Desnick et al.[60], revealed that as many as 20% of male and 25% of females with later onset Fabry mutations and hypertrophic cardiomyopathy concurrently have a pathogenic or likely pathogenic mutation of the HCM causing gene. Therefore, in addition to Gb3 accumulation, there should be some predisposing factors contributing to the pathogenesis of later-onset Fabry cardiomyopathy.

Regarding hyperlipidemia in Fabry patients, circulating Gb3 has been proved to be primary transported by LDL and HDL lipoproteins (approximately 60% and 30% respectively) [61]. Furthermore, hyperlipidemia was correlated with Fabry disease [62] and selective LDL-apheresis was shown to be an effective treatment in a Fabry patient with recurrent strokes [63]. However, the causal relationship between hyperlipidemia and hypertrophic cardiomyopathy of later-onset Fabry patients still needs to be clarified.

Interestingly, only scanty Gb3 accumulation could be found in the cardiomyocytes of our three patients who had received the longest period of ERT. This finding is quite different from several previous studies that showed the histological clearance of Gb3 accumulation from cardiomyocytes is usually poor [38, 49, 54, 64]. Does this finding indicate that a longer duration of ERT still has efficacy for Gb3 clearance from cardiomyocytes of later-onset Fabry patients? However, without pre-ERT biopsies, it is impossible to claim ERT was able to clear Gb3 accumulation from the cardiomyocytes of these three patients. More cardiac biopsy data with pre-ERT and post-ERT should be collected to clarify this point.

Recent studies revealed that gadolinium-contrast cardiac MRI is a good method to assess myocardial fibrosis in Fabry disease [34, 65‐67]. In patients without fibrosis, ERT resulted in a significant reduction in left ventricular mass and improvement of myocardial function. In contrast, patients with mild or severe fibrosis showed a minor reduction in left ventricular hypertrophy and no improvement in myocardial function. In this study, one patient was found to have significant interstitial fibrosis among his cardiomyocytes by H&E staining of endomyocardial biopsy. However, no significant fibrosis was observed in his heart with late-enhancement technique by gadolinium-contrast cardiac MRI examination. On the other hand, 6 out of the 20 patients received gadolinium-contrast cardiac MRI examination in this study were found to have severe fibrosis (Table 1), but none of them were found to have any significant cardiac fibrosis in their endomyocardial biopsies. This finding indicates that the histological fibrosis of interventricular septum from right ventricular endomyocardial biopsy does not reflect the severity of the fibrosis of the whole heart; most of the fibrosis that significantly affect heart function is usually located in the left ventricular area in Fabry disease.

In this study, we analyzed the pathologic appearance of cardiomyocytes by endomyocardial biopsies in 22 atypical later-onset Fabry disease patients with the Chinese mutation (IVS4 + 919G > A). These data suggest that IVS4 + 919G > A is a real pathogenic mutation with typical Gb3 accumulation and hypertrophic changes in cardiomyocytes. No endothelial Gb3 accumulation was found in cardiac biopsies is consistent with the cardiac pathologic findings of later-onset Fabry disease.

Fabry disease is a rare lysosomal disorder resulting from a genetic deficiency of α-Gal A. The present study used endomyocardial biopsies to confirm that the Chinese Fabry mutation (IVS4 + 919G > A) is a real pathogenic mutation.