Difficulties in Diagnosing Fabry Disease in Patients with Unexplained Left Ventricular Hypertrophy (LVH): Is the Novel GLA Gene Mutation a Pathogenic Mutation or Polymorphism?

The heritable lysosomal storage disorder Fabry disease (FD) occurs because of an α-galactosidase (α-GLA) enzyme deficiency that advances with glycosphingolipid metabolism disorder (1). Being an X-linked disease, FD can be transferred by males as well as females (2). Over 1,000 GLA gene variants have been discovered across the world. Fabry disease may be caused by a single nucleotide variation in the GLA (3). Due to a defect in the α-GLA enzyme, glycosphingolipids steadily accumulate in lysosomes, particularly globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), in different cell types within the body. This gives rise to multisystemic issues, such as ocular, neurological, renal, brain, skin, and cardiac symptoms (4).

Brain, renal, and cardiac activity play vital parts in diagnosing FD. Approximately 60% of patients with FD have some form of heart involvement (5). The accumulation of glycosphingolipids in myocytes causes hypertrophy and the eventual fibrosis of the myocardium (6). Although the actual prevalence of FD is unknown, it is estimated to range from 1:40,000 to 1:117,000 people (7). The actual incidence and prevalence are unknown due to the presence of atypical or oligosymptomatic forms (8). Patients with stroke, idiopathic renal failure, or cardiomyopathy are frequently screened for FD in screening studies (9). Cardiac involvement is the leading cause of death in patients with FD. For this reason, it is important to diagnose these patients early because the available treatments are more efficient when they are started before the progression of the disease (10, 11).

Enzyme replacement therapy (ERT) and pharmacological chaperones can be used in the treatment of FD. Since the most common finding in patients with cardiac involvement is left ventricular hypertrophy (LVH), LVH has been evaluated in terms of FD in patients with unexplained LVH in screening studies (12). In these studies, the prevalence of FD varies according to the subject country and the screening methods used. In addition to known genetic mutations associated with FD, new genetic mutations or polymorphisms associated with the disease can be detected in screening studies.

This study aimed to present screening results for FD in patients with unexplained LVH diagnosed by two-dimensional echocardiography.

Over 120 patients older than 30 years who were diagnosed with unexplained left ventricular (LV) wall thickness of ≥ 13 mm through echocardiography were screened for FD between March 2020 and March 2021. Patients were excluded who were suffering from major heart valve disease, significant hypertension, coarctation, strain conditions like aortic stenosis, earlier diagnosis of FD, previous history of any disease that was linked to LVH, or a family history of autosomal dominant hypertrophic cardiomyopathy (HCM) or FD. All participants provided their written informed consent. The study was conducted in conformance to the Helsinki Protocol and obtained the approval of the local ethics committee.

The collection of peripheral venous blood samples from all the participants of the study was carried out in the EDTA tubes (2 ml) for performing mutation analysis. These samples were sent to a laboratory specialized in diagnosing genetic disorders. In all patients, GLA gene sequence analysis was carried out. If a GLA gene mutation was identified, measurements for α-Gal A enzyme activity and lyso-Gb3 levels were obtained. In patients with gene mutations, a family screening was also carried out.

The clinical and demographic characteristics and the echocardiographic measurements of 120 patients with idiopathic LVH who underwent genetic examination are shown in Table 1. The majority of the patients were female (n = 58, 69%), and the mean age was 60.3 ± 15.7 years. The mean echocardiographic parameters were as follows: LVEF: 61.4% ± 4.8%, IVS: thickness 15.2 ± 5.4 mm, LVPWD: 13.5 ± 2.1 mm, LVEDD: 50.4 ± 5.2 mm, LVESD: 30.8 ± 8.5 mm, and left atrial diameter: 44.5 ± 6.5 mm. GLA gene mutations were detected in three male patients. One of these mutations (NM_000169.2:IVS6-10G>A [c.1000-10G>A]) was previously associated with FD. Another mutation’s association with FD was considered a benign GLA variant (NM_000169.2:c.937G>T [p.D313Y] [p.Asp313Tyr]). A third was a mutation that had not been previously associated with FD (NM_000169.2:c.941A>T [p.K314M] [p.Lys314Met]). The three mutations mentioned are discussed below with case examples.

Case 1

This 56-year-old male patient presented to cardiology with dyspnea on exertion and palpitations. His systemic arterial pressure was 135/82 mmHg, and his heart rate was arrhythmic at 110 beats per minute. His electrocardiogram demonstrated atrial fibrillation and LVH, and his TTE showed concentric LVH, with an EF of 65%. Cerebral white matter lesions were detected on cranial magnetic resonance imaging, and microalbuminuria was detected in a urine analysis. The patient had low α-Gal A activity at 2.80 nmol/mg/h (normal range: >23.10 nmol/mg/h), and his lyso-Gb3 level was high at 12.80 ng/mL (normal range: <1.30). A genetic analysis revealed the NM_000169.2:IVS6-10G>A (c.1000-10G>A) mutation, which was consistent with the diagnosis of FD (14) (Figure 1). The patient’s family screening showed a similar mutation in seven relatives. For the index patient, ERT was started.

Figure 1.

Case 2

This 58-year-old male patient, who was experiencing chest pain and shortness of breath, presented to the cardiology department. The patient was suffering from coronary artery disease and hypertension and had undergone stunting in the right coronary artery and the left anterior descending coronary artery. A genetic assessment was performed because of unexplained concentric LVH on echocardiography, and the NM_000169.2:c.937G>T (p.D313Y) (p.Asp313Tyr) mutation was found (15) (Figure 2). The patient had normal α-Gal A activity and normal lyso-Gb3 levels. His nephrological, neurological, and ocular assessments were also found to be normal.

Figure 2.

Case 3

This 58-year-old male patient presented to cardiology with palpitations. He had no known cardiac history. His electrocardiography results showed a normal sinus rhythm and concentric LVH, and his neurological, nephrological, ocular, and other systemic examinations were normal. A genetic analysis revealed the NM_000169.2:c.941A>T (p.K314M) (p.Lys314Met) mutation (14) (Figure 3). The patient had normal α-Gal A activity and normal lyso-Gb3 levels. The same genetic mutation was discovered in two siblings during family screening. However, the patient’s echocardiographic examinations were normal.

Figure 3.

In this study, 120 patients with idiopathic LVH on echocardiography were genetically screened for FD. A genetic mutation was detected that was previously associated with FD in one of the participants (Case 1). The D313Y mutation was detected in another participant (Case 2). This mutation was previously described as a benign GLA gene mutation. In the third participant (Case 3), a result was obtained that is considered a polymorphism in the foreground.

Variants in the α-GLA A gene present on the X chromosome cause FD (1). Due to a deficiency in the α-Gal A enzyme, the glycolipid Gb3 steadily accumulates within lysosomes. The degree of X inactivation determines the intensity of FD in females (4). Multisystemic involvement is exhibited in FD (16). It is evident from the recurring observation of cardiac involvement in FD that the heart is extremely sensitive to α-GLA A (17, 18) and that cardiac involvement is the main cause of morbidity and mortality in patients with FD (10).

Differences in the incidence and occurrence of FD signify variations in study design and population. There is a low prevalence of the disease in the general population; however, it is increasing in different at-risk patient populations, for example, those with kidney failure, stroke, or HCM (12). Nakao et al. determined seven patients to be suffering from FD out of a group of 230 Japanese males with LVH (12). Sachdev et al. identified six patients as having FD out of 153 males with HCM (4%) in the United Kingdom, with a prevalence of 6% for males diagnosed at over 40 years of age (19). Ommen et al. determined that out of 100 patients with HCM in the United States who had undergone septal ablation, none had FD (20). Chimenti et al. determined four Italian patients to have FD out of a total of 34 females (12%) with HCM who had undergone endomyocardial biopsy (17). Monserrat et al. screened plasma for α-Gal A activity in a Spanish population of patients with HCM and determined an FD prevalence of almost 2% (0.9% in males and 1.1% in females). However, this study included many variants of unknown significance (21). Havndrup et al. identified three patients with FD from a group of 90 patients with LVH in Denmark (18). Elliot et al. identified seven patients with FD from a group of 1,386 European patients (3.4%) with HCM or unexplained LVH (22). Hagege et al. performed systematic screening for FD (an α-Gal A assay on dried blood spots via a filter paper test) in a French population of patients diagnosed with HCM and found four patients with FD from a total of 278 males (23). However, Mawatari found no FD in a Japanese population of 738 patients with unexplained LVH (24). Five patients with FD were identified by Terryn et al. from a group of 560 Belgian patients with unexplained LVH (25). Out of a group of 100 Czech patients, Palecek et al. found four patients with FD (26). Baptista et al. performed screening for FD in Portuguese patients with LVH, and a single case of FD was identified from a total of 47 patients (27). A Spanish group of 805 patients with clinical symptoms related to FD was examined by Vieitez et al., and 21 patients were found to have FD (28). Maron et al. performed screening for FD among patients from North America with diagnosed HCM. The study found two patients with FD from a total of 585 patients (29). From a Korean group of 988 patients with LVH, Kim et al. found five patients with FD (30). Similarly, in our study, two patients out of 120 with unexplained LVH were diagnosed with FD by genetic analysis (1.6%) (Figure 4). In these screening studies, different LVH cut-off values were used. Similarly, the diagnostic methods for FD varied in these studies.

Figure 4.

The D313Y genotype, previously described as a benign mutation not clinically relevant to FD (15), was reported in 2016 to have a significant impact on health-related quality of life of respective individual patients. This mutation might represent a confounding risk factor for certain isolated symptoms, triggering a specific mild clinical variant of FD (31).

During these screening studies, some of the genetic mutations detected by genetic analyses were pathogenic, while some were polymorphisms. For instance, Montserrat et al. reported an incidence of 1% in the Spanish population but includes many variants of unknown significance (21). The difficulty lies in identifying GLA variants that may give rise to the clinical symptoms of FD. There is a lack of enzyme activity in patients with classic FD who have clinically significant α-GLA A enzyme deficiency (32). It was found that the residual activity of 30% to 35% of the mean normal α-GLA A activity was the threshold for diagnosing FD (33).

There are three categories of α-GLA A residual activity into which GLA variants are grouped. In the first category, patients have benign GLA gene mutations and a 10% change in enzyme levels (34). In the second category, enzyme activity is between 15% to 30% of the standard activity in males (35). The clinical expression of the second group is highly dependent on genetic and epigenetic modifiers. In the third category, there is considerably reduced enzyme activity (GLA variants less than 35% to 40% of the average normal male controls). The true pathogenicity threshold for FD in terms of enzyme levels is unknown.

Normal enzyme levels in male patients indicate benign GLA variants and polymorphisms in these patients. Since the enzyme levels of the patients in Case 2 and Case 3 were normal, the genetic result was evaluated as a predominantly benign GLA variant and polymorphism. Their cardiomyopathy likely has another etiology, and the variant found was a benign polymorphism. These results demonstrate that enzyme activity levels alone are insufficient to determine whether a particular GLA variant is pathogenic. In addition to enzyme levels, the accumulation of substrate in plasma or urine or altered sphingolipid levels (Gb3 or lyso-Gb3) may be helpful in the diagnosis of FD (14, 15). However, recognizing typical lysosomal inclusions in tissue biopsy specimens is suggested as the best method of diagnosing FD (36).

Fabry disorder is rare in that it is one of the metabolic diseases that can be treated. As it is a progressive disease, the early diagnosis of FD is vital, and it can be treated with ERT and pharmacological chaperones (37). If FD is diagnosed via screening, it is extremely important to screen family members. Some individuals may be identified via family screening who have not yet developed organ damage (with the exception of the index case).

In conclusion, early diagnosis is of vital importance in FD, which can be treated with enzyme replacement. Patients over 30 years of age with idiopathic LVH should be screened for FD because a patient with unexplained LVH is part of a high-risk group that has a high incidence of FD. In screening studies, clinical evaluation of organ involvement as well as enzyme and lyso-Gb3 levels are required to show whether the newly detected mutations are true pathogenetic mutations or polymorphisms. Identifying new polymorphisms is important to know the true mutations in FD.