Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Massive accumulation of globotriaosylceramide in various tissues from a Fabry patient with a high antibody titer against alpha-galactosidase A after 6 years of enzyme replacement therapy.

Fabry disease is an X-linked metabolic disorder due to a pathogenic mutation of the GLA gene. The accumulation of globotriaosylceramide (Gb3) damages multiple organs, including the heart, kidney and nervous system, especially in classical type Fabry disease. Enzyme replacement therapy (ERT) using recombinant alpha-galactosidase A has been shown to remove Gb3 from organs and to improve the prognosis of Fabry disease. We herein report the case of a 67-year-old classical type Fabry patient who had been treated with ERT for 6 years and who continuously showed a high antibody titer against recombinant alpha-galactosidase A during therapy. A post-mortem examination was performed after sudden death. A histological examination revealed the massive accumulation of Gb3 in various organs, even after long term ERT. In addition to the typical pathological findings as reported in tissue biopsy samples, the serious accumulation of Gb3 in the cardiac conduction system and the endocrine system was detected. Since the start of ERT for this patient might be too late to improve organ damage and prognosis, ERT should be started before the appearance of major organ involvement for the effective elimination of Gb3 and changes in the therapeutic strategy might be considered if the patient shows a high antibody titer against recombinant alpha-galactosidase A.

Clinical findings of gadolinium-enhanced cardiac magnetic resonance in Fabry patients.

BACKGROUND: Fabry disease is one of the causes of left ventricular hypertrophy (LVH) and can be treated with enzyme replacement therapy or pharmacological chaperone therapy. Late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) can identify myocardial fibrosis and be used for the stratification in LVH. However, the details of the prevalence and characteristics of LGE in Japanese Fabry patients have not been reported. METHODS: We evaluated myocardial involvement in 26 Fabry patients (10 males, 16 females) using gadolinium-enhanced CMR. LGE areas were analyzed using the previously reported scoring method. Echocardiography was also performed to evaluate the left ventricular function and left ventricular mass. RESULTS: LGE on CMR images was positive in 5 out of 26 patients, and all patients with LGE-positive findings suffered from LVH (2 out of 5 male patients and 3 out of 4 female patients with LVH on echocardiography). LGE was specifically localized at the mid-wall in the infero-lateral area of the left ventricle. LGE-positive patients seemed to be older, and tended to have a larger left ventricular mass index and higher B-type natriuretic peptide level than LGE-negative patients. CONCLUSIONS: These results revealed that specific localization of LGE was present in Fabry patients.

Mutation spectrum of α-Galactosidase gene in Japanese patients with Fabry disease.

The efficacy of pharmacological chaperone therapy for Fabry disease depends on the type of α-galactosidase A (GLA) mutations. Here, we examined the mutation spectrum of the GLA gene among patients from 115 Japanese families with Fabry disease. Of these, no pathogenic mutations were identified in six families (5.2%). In total, 73 different disease-causing mutations were identified: 41 missense (56.2%), 11 nonsense (15.1%), four in frame deletion (5.5%), 10 frameshift (13.7%), six splice site (8.2%), and one intronic (1.4%) mutations. The GLA mutations detected in later-onset phenotype patients with end-stage renal disease overlapped with those seen in classical patients, indicating that it is difficult to differentiate between these two phenotypes from gene mutations. Additionally, 33 families (28.7%) had amenable mutations to the pharmacological chaperone migalastat. In conclusion, our study is informative when considering genetic counseling and pharmacological chaperon therapy for Fabry disease.

Biallelic mutations of EGFR in a compound heterozygous state cause ectodermal dysplasia with severe skin defects and gastrointestinal dysfunction.

Epidermal growth factor receptor (EGFR), a receptor that recognizes epidermal growth factor, is a very important regulator of cell proliferation and differentiation. To date, three cases of severe ectodermal dysplasia were reported to be caused by an inherited germline homozygous loss-of-function missense mutation of EGFR. This is the first report of a patient with biallelic compound heterozygous mutations in EGFR.

The beneficial effects of long-term enzyme replacement therapy on cardiac involvement in Japanese Fabry patients.

Fabry disease is a hereditary disorder that occurs due to the reduction or absence of alpha-galactosidase A activity, which leads to cardiac involvement including left ventricular hypertrophy (LVH). Enzyme replacement therapy (ERT) provides better patient outcomes by preventing serious complications. However, there have been very few studies on the long-term effects of ERT on the cardiac manifestations in Japanese Fabry patients. We retrospectively analyzed the data from the medical records of 42 Fabry patients (male, n = 17; female, n = 25) who were followed at Jikei University Hospital, and in whom the long-term effects of ERT could be evaluated (median follow-up period: male, 11 years; female, 8 years). The slope of the left ventricular mass (LVM) increase was 3.02 ±â€¯3.41 g/m2/year in males and 1.69 ±â€¯2.73 g/m2/year in females. In a subgroup analysis, the slopes of males with and without LVH did not differ to a statistically significant extent; however, the slope in female patients without LVH was significantly smaller than that of female patients with LVH. We then compared our data to the natural historical data that have previously been reported. In comparison to the previously reported data, we found a significant reduction in the LVM changes (g/height2.7/year) of patients who received long-term ERT (male, 4.07 ±â€¯1.03 to 1.25 ±â€¯1.39; female, 2.31 ±â€¯0.81 to 0.78 ±â€¯1.23). Long-term ERT effectively prevents LVH in Fabry patients. This effect was also observed in the patients with LVH prior to the initiation of ERT.

Characteristics of Cerebral Microbleeds in Patients with Fabry Disease.

BACKGROUND AND PURPOSE: Fabry disease (FD) is an X-linked lysosomal storage disorder frequently associated with the central nervous system manifestations. Although white matter hyperintensity (WMH) on MRI has been previously reported, little is known about cerebral microbleeds (CMBs) in patients with FD. Our aim is to investigate the clinical characteristics of CMBs in patients with FD. METHODS: All patients with FD were diagnosed by enzyme activity and/or gene analysis at Jikei University Hospital. We retrospectively enrolled consecutive patients with FD who underwent MRI study, including fluid-attenuated inversion recovery and susceptibility-weighted imaging, between July 2008 and September 2013. After categorizing the patients into CMB-positive and CMB-negative groups, we compared the clinical characteristics between the 2 groups. RESULTS: We enrolled 54 patients (males, 24; median age 39 years, interquartile range; 29-50 years). The CMB-positive group included 16 (30%) patients. The number of males was significantly higher in the CMB-positive group than in the CMB-negative group (75% versus 32%, P = .003). The prevalence rates of chronic kidney disease (CKD) (estimated glomerular filtration rate < 60 mL/min/1.73 m(2)) and WMH were higher in the CMB-positive group than in the CMB-negative group (CKD: 44% versus 13%, P = .013; WMH: 88% versus 58%, P = .035). No significant differences in the number of vascular risk factors were observed between the 2 groups. CONCLUSIONS: The distinct characteristics of FD patients with CMBs were male sex, presence of CKD, and WMH. These factors may play an important role in the mechanism of hemorrhagic stroke in FD.

Prevalence of Fabry disease in dialysis patients: Japan Fabry disease screening study (J-FAST).

BACKGROUND: In Fabry disease, progressive glycolipid accumulation leads to damage in kidney and other organs. This study was designed to determine the prevalence rate of Fabry disease in Japanese dialysis patients. METHODS: All dialysis patients agreeing to Japan Fabry disease screening study (J-FAST) with informed consent were selected except for Fabry disease. The screening was performed by a method of measuring plasma and/or leukocytes lysosomal α-galactosidase A protein level and α-galactosidase A activity. If positive, genetic analysis was carried out upon patient's agreement. RESULTS: J-FAST dealt with 8547 patients (male 5408, female 3139). At the tertiary examination, 26 out of 8547 patients were found to be positive. Six out of 26 patients could not accept genetic analysis because of death. Remaining 20 patients agreed with genetic analysis; then 2 patients (male 2, female 0) had a variation of the α-Gal gene and 11 patients showed E66Q variations. Therefore, the frequency of Fabry disease in J-FAST was 0.04 % (2/5408) in males and 0 % (0/3139) in females, and then 0.02 % (2/8547) in all patients. The presumptive clinical diagnoses of end-stage kidney disease (ESKD) were 10 chronic glomerulonephritis, 7 diabetic nephropathy, 3 unknown etiology, 3 nephrosclerosis, 1 gouty nephropathy, 1 autosomal dominant polycystic kidney disease and 1 renal tuberculosis among 26 tertiary positive patients. Two male Fabry patients were initially diagnosed as nephrosclerosis and chronic glomerulonephritis. CONCLUSIONS: The prevalence rate of Fabry disease in J-FAST was 0.02 %. Moreover, Fabry disease could not be ruled out as the clinical diagnosis of ESKD.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Enzyme replacement therapy in two Japanese siblings with Fabry disease, and its effectiveness on angiokeratoma and neuropathic pain.

Enzyme replacement therapy (ERT) for Fabry disease does not show a clear benefit in angiokeratoma. We describe two Japanese siblings with Fabry disease, who were diagnosed when angiokeratomas were found on the older sibling at the age of 13 years. Neither of the boys complained of pain, while both suffered from hypohidrosis. We evaluated the safety and efficacy of ERT with recombinant human agalsidase alfa (Replagal®, Dainippon-Sumitomo Pharma. Co., Osaka, Japan) in these siblings over a 5-year period. In both siblings, sweating was observed 3 months after the initiation of ERT, which motivated them to adhere to ERT. Pain sensation was regained after 12 to 36 months of ERT, followed by a decrease after 48 to 60 months. Angiokeratomas on the lateral side of the knee of the older sibling partially disappeared after 48 months of ERT. Although the height of both siblings at baseline was lower than the corresponding average age-related heights in the normal Japanese population, during ERT they were within, or close to, the average +1 standard deviation in the non-Fabry population. Their growth rate seemed to indicate catch-up growth. Other clinical symptoms were maintained at baseline levels. Immunoglobulin G anti-agalsidase alfa antibodies were not detected in both sibling during ERT, and no infusion-associated reaction was observed. The treatment was generally well tolerated. ERT was a safe and effective treatment for angiokeratoma and neuropathic pain for these two siblings with Fabry disease.

No accumulation of globotriaosylceramide in the heart of a patient with the E66Q mutation in the α-galactosidase A gene.

BACKGROUND: Fabry disease is an X-linked lysosomal disorder resulting from mutations in the α-galactosidase A (GLA) gene. Recent reports described that the E66Q mutation of GLA is not a disease-causing mutation. However, no pathological study was reported. We carried out pathological studies using a cardiac biopsy specimen from a patient with the E66Q mutation. MATERIALS AND METHODS: The case was a 34 year old male patient with end-stage renal failure and cardiomegaly. He was diagnosed with gout at 15 years of age and hemodialysis was started for gouty nephropathy from 31 years of age. He was suspected of having Fabry disease as the result of a screening study for Fabry disease in patients with end-stage renal failure and was referred to our hospital for mutation analysis of the GLA gene. We carried out enzymatic and genetic analysis for GLA and pathological studies of a cardiac biopsy specimen. RESULTS: The patient had the E66Q mutation in the GLA gene. GLA activity in leukocytes was 36.2% of the average of normal controls. The pathological study of the cardiac biopsy sample showed no characteristic findings of Fabry disease. The immunohistochemistry for GL3 of the cardiac biopsy sample showed no positive cells. CONCLUSION: Although the E66Q mutation reduced enzyme activity, the characteristic pathological findings of Fabry disease and the abnormal accumulation of GL3 were not detected in cardiac tissues. The E66Q mutation of the GLA gene is thought to be a functional polymorphism based on enzymatic and pathological studies.

Myeloschisis repair in a premature neonate with a birth weight of 599 g.

This report describes the case of a neonate with myeloschisis weighing 599 g that underwent an operation for spinal cord reconstruction resulting in substantial neuronal functional recovery. At 28 weeks of gestation, oligohydramnios was detected and the female fetus was diagnosed with intrauterine growth retardation. At birth by emergency caesarean section, she showed lumbosacral myeloschisis and complete paralysis of the lower extremities. Surgical repair and spinal cord reconstruction was performed 2 days after birth by closing the neural placode. Total blood loss was only 2 ml. Within 3 months of the operation, lower extremity neurologic function gradually improved down to the level of the knee joint. To our knowledge, this case is the lowest recorded body weight for a neonate with myeloschisis repair at birth, and this further suggests the possibility of improvement of lower extremity neurologic function after birth and surgical reconstruction.

Coincidental finding of Fabry's disease in a patient with IgA nephropathy.

We present the case of a woman with IgA nephropathy and concomitant Fabry's disease. She was referred to our hospital with proteinuria and haematuria. A renal biopsy showed findings indicating IgA nephropathy under light and immunofluorescence microscopy. Electron microscopy, however, showed laminated inclusion bodies characteristic of Fabry's disease. The α-galactosidase activity in her serum was low, and the diagnosis of Fabry's disease was confirmed by genetic analysis. Fabry's disease in a patient with IgA nephropathy is a very rare occurrence, and Fabry's disease diagnosed only by electron microscopy has not been previously reported.

Non-invasive high-risk screening for Fabry disease hemizygotes and heterozygotes.

The most appropriate time for screening for Fabry disease (FD) is school age. For this reason, we developed non-invasive methods for measuring urinary alpha-galactosidase A (alpha-gal A) protein, using enzyme-linked immunosorbent assay (ELISA), and for globotriaosylceramide (GL-3), using tandem mass spectrometry (MS/MS). We measured these two biomarkers in the urine of previously diagnosed FD hemizygotes and heterozygotes, and in controls. All the classic FD hemizygotes were clearly distinguished from controls by either method alone, and combining the two assays produced 96% sensitivity for detecting heterozygotes. To assess the utility of these methods for screening school children and adults at high risk of FD, a pilot study was conducted. To distinguish FD from 432 controls, cut-off values for alpha-gal A protein and GL-3 were set at the 5th and 95th centile values of the controls, respectively. Among the high-risk patients, the measurements exceeded the cut-off values for both biomarkers in male and female subjects and were strong indicators for Fabry hemizygotes and heterozygotes. However, we recommend that if the results of the first measurements exceed the cut-off values for only one of these biomarkers, another urine sample should be requested for re-assay to confirm the result.

Significance of screening for Fabry disease among male dialysis patients.

BACKGROUND: Fabry disease is an X-linked disorder resulting from a deficiency of the lysosomal enzyme alpha-galactosidase A(alpha-Gal A). Renal insufficiency is a very important manifestation and affects the prognosis of patients. Recently, a renal variant type that is characterized by low plasma alpha-Gal A activity and a milder phenotype, but which progresses to end-stage renal failure, has been reported. In this study, we clarified the incidence of this atypical variant of Fabry disease in hemodialysis patients. METHODS: We measured plasma alpha-Gal A activity in 450 male dialysis patients who had never been diagnosed with Fabry disease. RESULTS: The mean of the alpha-Gal A activity of the patients was 9.75 +/- 3.20 nmol/h/ml, while the controls with classical Fabry (n = 3) were 0.52-1.04 nmol/h/ml. Among the patients, one patient was found to exhibit low alpha-Gal A activity in plasma (3.18 nmol/h/ml) and in leukocytes (0.639 nmol/h/mg). This patient was a 43-year-old Japanese man who had been on regular dialysis since the age of 23. He did not present typical clinical signs of classical Fabry, such as acroparesthesias or hypohidrosis, but did present renal insufficiency and severe left ventricular hypertrophy which had developed only recently, suggesting a variant form of Fabry disease. Sequencing of the DNA of this patient revealed a deletion of a single amino acid of valine in 10252. CONCLUSIONS: A case of an atypical variant of Fabry among 450 male dialysis patients (0.22%) was found in the survey. This indicates the potential for undiagnosed Fabry disease among dialysis patients. The results of this study indicate the significance of screening for Fabry disease among male dialysis patients.

Non-invasive screening method for Fabry disease by measuring globotriaosylceramide in whole urine samples using tandem mass spectrometry.

Fabry disease is an X-linked sphingolipidosis due to a deficiency of alpha-galactosidase A, which leads to the accumulation of globotriaosylceramide (GL-3) in several organs. When recombinant human alpha-galactosidase A is intravenously administered repeatedly before the patient develops permanent tissue damage, there is evidence that the accumulation of GL-3 is decreased in some organs and that the clinical symptoms are alleviated in some patients. However, Fabry disease is rare and many patients are not diagnosed until adulthood after irreversible tissue damage has occurred. Our group has developed a simple and non-invasive screening method for Fabry disease that measures total GL-3 in whole urine samples by tandem mass spectrometry. Using this method, we found that the concentration of GL-3 in whole urine sample from hemizygous patients, including pre-symptomatic young children with classic type Fabry disease, was significantly higher than that in controls. The mean concentration of GL-3 in urine from heterozygotes with symptoms was significantly higher than control concentrations, but GL-3 levels in the urine from 2 out of 8 heterozygotes of classic type Fabry disease were within control levels. An asymptomatic 14-year old hemizygote in the family of a cardiac variant did not have elevated urinary GL-3. Therefore, screening for the classic type and probably renal variant of Fabry disease is possible by measuring urinary GL-3, using our method. The early diagnosis of cardiac variant hemizygotes and some heterozygotes with all types of Fabry disease will not be possible using our method. We propose that this procedure can be used as a reliable, non-invasive, simple method for general and high-risk population screening for hemizygotic patients with the classic type and probably renal variant of Fabry disease.