Identification of a novel loss-of-function mutation of the GLA gene in a Chinese Han family with Fabry disease

We report a four-generation family comprising 51 members (28 males, 23 females, 4 people of them died). The proband was a 65-year-old male patient. Seven of his family members were also included in this study. The patient underwent thorough physical examinations and other tests, including urine protein tests, electrocardiography, echocardiography, cardiac magnetic resonance, α-Gal A enzyme activity testing, genetic testing and skin biopsy. The family members were subjected only to a review of their medical histories, physical examination and genetic testing. The clinical features of the other patients in this pedigree were dictated by the proband, and the family members were enrolled in the present study. A total of 300 ethnically matched controls were used to detect the allele frequency of the identified variant in the normal population. Written information consent for participation and publication were obtained from all the individuals which are described in this manuscript. The patient in Fig. 2a consented to the inclusion of his picture in this manuscript in written format. The parents provided consent in the case of children under 16. The present study was approved by the Medical Ethics Committee of Tongji Hospital, Tongji Medical Collage, Huazhong University of Science and Technology, and complied with the principles of the Declaration of Helsinki.

Peripheral blood samples were collected from the patient, seven of his family members, and normal controls by using EDTA-K2 tubes. Genomic DNA was extracted by using the AP-96-BL-GDNA-12 kit (AXYGEN BIOSCIENCES, California, USA) according to the manufacturer’s instructions and stored at − 80 °C until further use. All seven exons and adjacent noncoding regions were amplified by PCR and subsequently screened via directional Sanger sequencing with the ABI 3130 BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3130xl sequencer. The primer sequences and amplicon sizes are shown in Table 1. The sequence analyses were performed by using a 3130 Genetic Analyzer (Applied Biosystems, USA). Genetic screenings of the mutation sites were conducted for 300 local control subjects.

Bioinformatics analysis of the detected mutation was performed using Polyphen and SIFT software. The PolyPhen2 score ranges from 0 to 1, and the corresponding predictions are “probably damaging” (score > 0.85), “possibly damaging” (0.15 < score < 0.85), and “benign” (score < 0.15). If the SIFT score is smaller than 0.05, then the corresponding neutral substitutions (NS) are predicted to be “damaging”; if the SIFT score is greater than 0.05, then the NS are predicted as “tolerated”. The variant with a higher score is considered more deleterious in Phylop software.

Human alpha-Gal A (NM_000169.2) was amplified and cloned into the mammalian expression vector pcDNA3.1 (+) by introducing unique restriction sites for Hind III and Not I. The QuikChange II site-directed mutagenesis kit (Agilent Technologies, USA) was used for site-directed mutagenesis to generate a single nucleotide change (Asn278Lys, A to T) in the GLA gene sequence. Expression vectors harbouring the wild type (GLA-278Asn) or mutant (GLA-278Lys) sequences were identified by Sanger Sequencing.

HEK 293 T cells were cultured in 10% FBS high-glucose DMEM at 37 °C with 5% CO₂ in a humidified incubator and seeded onto 24-well plates the day before transfection. Transient expression of the wild type and mutant enzymes was performed using Lipofectamine™ 2000 reagent (Invitrogen, USA) and antibiotic-free Opti-MEM® I Reduced Serum Medium (Gibco, Life Technologies, USA). After incubation for 4 h at 37 °C, the medium in the 24-well plates was replaced with 500 μl of fresh 10% FBS DMEM.

The enzyme activities of wild type and mutant α-Gal A were also investigated as previously reported [18, 19]. Briefly, the enzyme activity was calculated at pH 4.6 with 2 mM 4-MU-α-Gal (Sigma, Germany) as a substrate. The release of 4-MU per hour was determined by fluorescence measurement with an excitation wavelength of 360 nm and emission wavelength of 465 nm.

The proband (II: 3), a 65-year-old male patient, was admitted as a cardiovascular inpatient 5 years ago due to chest tightness, dyspnoea (New York Heart Association functional class III) and suspected primary senile degenerative heart disease (Fig. 1). During the past 5 years, the patient received drug treatments, including vasodilators, diuretics, and cardiotonics, and the symptoms were slightly alleviated. Recalling his disease history, the patient displayed a three-year history of cerebral infarction, a one-year history of atrial fibrillation, a one-year history of thyroid dysfunction, and idiopathic thrombocytopenia for decades with several years of chronic diarrhoea. The patient also complained of frequent acroparaesthesia and hypohidrosis. Within 10 days prior to his visit to the hospital, the patient showed symptoms such as loss of appetite, bloating, abdominal pain, and vomiting after eating with no obvious trigger. The patient denied histories of hypertension, diabetes, and any infectious diseases. Physical examination of his skin showed a large area of skin rash that was particularly prominent in the groin and on the back, which were identified as angiokeratomas by histopathology (Fig. 2a). Blood tests showed mild anaemia (haemoglobin: 114 g/l), decreased eGFR (74.4 ml/min/1.73m²), an increased level of NT-proBNP (3904 pg/ml), and slightly elevated levels of troponin (0.046 ng/ml). Urine test results showed that the patient had proteinuria (1+). The electrocardiogram showed atrial fibrillation, voltage criteria for LV hypertrophy, and T wave inversion in all precordial leads (Fig. 2b). Transthoracic echocardiography demonstrated concentric LV hypertrophy (14 mm for the interventricular septum and 13 mm for the left ventricular posterior wall) and enlargement of the double atriums, consistent with signs detected by cardiac magnetic resonance imaging (Fig. 2c). Importantly, the enzyme activity of α-Gal A measured in the proband was very low, at 0.85 nmol/h/mg (> 30 nmol/h/mg for a normal person). A review of his family history revealed that two of his siblings (II: 2, female; and II: 5, male) presented renal dysfunction as the most severe manifestation and died from uraemia several years ago. His three surviving siblings (II: 7; II: 10; and II: 11), his two daughters (III: 12 and III: 14), nine of his nephews and nieces (III: 2; III: 8; III: 16; III: 18; III: 20: III: 22: III: 23: III: 24; and III: 26), and one of his grandsons (IV: 5) also had varying degrees of acroparaesthesia, hypohidrosis and chronic renal dysfunction. The male relatives exhibited more severe phenotypes than the female relatives. The clinical details of the subjects enrolled in this study are summarized in Table 2. The proband was treated based on the updated guidelines of heart failure. Specifically, vasodilators, diuretics, and digoxin were used to ameliorate the symptoms of heart failure, and a beta-blocker was used to improve the long-term prognosis. Warfarin was used to prevent stroke caused by atrial fibrillation. In addition, their families received genetic counselling in the genetic counselling centre of our hospital.

Detailed physical examination and laboratory inspections strongly conflicted with the previous diagnosis and suggested FD. To further clarify the diagnosis, we screened the entire coding region of the GLA gene for the proband of this pedigree and detected no mutations, except for p. Asn278Lys (c. 834 T > A) in exon 6 (Fig. 3a). We obtained blood samples from his three siblings (II-7, II-10 and II-11), two daughters (III-12 and III-14) and two grandsons (IV-5 and IV-6) and genotyped this mutation site by sequencing. The hemizygous p. Asn278Lys mutation was identified in the 3 male patients (II-7, II-11 and IV-5), and the heterozygous p. Asn278Lys mutation was identified in the 3 female patients (II-10, III-12, and III-14) in this pedigree. IV-6 (grandson of the proband) showed no abnormal clinical manifestations and was shown to be wild type at this site by sequencing.

The Genome Browser database (http://​genome.​ucsc.​edu) of vertebrate species revealed a strictly conserved genomic structure around the mutation site among vertebrate species (Fig. 3b). The function of the novel c.834 T > A mutation (p. Asn278Lys) was evaluated in silico by 4 independent software programs at the protein level. This mutation is predicted to be “possibly damaging” with a score of 0.755 by Polyphen-2. MutationTaster software annotated this mutation as “disease-causing” with a prediction score of 94. SIFT predicted this variant as a “damaging variant” (SIFT score = 0.005), while Phylop treated this mutation as a neutral variant with a score of − 0.99 (Fig. 3c). In addition, this mutation was shown to be absent in a control cohort of 300 local people.

To further identify the biological characteristics of the mutant enzyme in terms of protein degradation and activity, we developed an over-expression system in HEK-293 T cells to investigate the in vitro characteristics of the wild and mutant enzymes. When 293 T cells were transfected with GLA-Asn278 or GLA-Lys278, we obtained successful α-Gal A expression compared with the group transfected with the empty pcDNA3.1(+) vector. However, the mutant α-Gal A protein exhibited lower levels than the wild type protein (Fig. 3d). Similar results were observed for the residual activity of the mutant enzyme. As shown in Fig. 3e, the enzyme activity of α-Gal A was significantly lower in cells transfected with GLA-Lys278 compared with GLA-Asn278 (Fig. 3e).

To evaluate the therapeutic effect of DGJ as a potential drug for this particular mutation, we analysed the protein expression of α-GLA and enzyme activity changes after DGJ treatment. The results showed that the pharmacological chaperone DGJ increased the expression of α-Gal (Fig. 3d) and markedly normalized the activity of this enzyme (Fig. 3e), especially in cells expressing GLA-Lys278, which indicated that patients possessing this specific mutation were good responders to the potential biochemical therapy.