Analysis of caregiver perspectives on patients with mucopolysaccharidosis II treated with pabinafusp alfa: results of qualitative interviews in Japan.

BACKGROUND: Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked metabolic disorder predominantly affecting males. Pabinafusp alfa, an iduronate-2-sulfatase enzyme designed to cross the blood-brain barrier, was approved in Japan in 2021 as the first enzyme replacement therapy targeting both the neuropathic and somatic signs and symptoms of MPS II. This study reports caregivers' experiences of MPS II patients receiving pabinafusp alfa through qualitative interviews. METHODS: Semi-structured, qualitative interviews were conducted with caregivers at seven clinical sites in Japan using a semi-structured moderation guide (Voice of the Caregiver guide). Thematic analysis was applied to the interview transcripts to identify symptoms and health-related quality of life impacts at baseline, changes during treatment, and overall treatment experience. RESULTS: Seven caregivers from 16 trial sites participated, representing seven children aged 8-18 years who had received pabinafusp alfa for 3.3-3.5 years at the time of the interviews. Data suggest a general trend toward improvement in multiple aspects, although not all caregivers observed discernible changes. Reported cognitive improvements included language skills, concentration, self-control, eye contact, mental clarity, concept understanding, following instructions, and expressing personal needs. Further changes were reported that included musculoskeletal improvements and such somatic changes as motor function, mobility, organ involvement, joint mobility, sleep patterns, and fatigue. Four caregivers reported improvements in family quality of life, five expressed treatment satisfaction, and all seven indicated a strong willingness to continue treatment of their children with pabinafusp alfa. CONCLUSION: Caregivers' perspectives in this study demonstrate treatment satisfaction and improvement in various aspects of quality of life following therapy with pabinafusp alfa. These findings enhance understanding of pabinafusp alfa's potential benefits in treating MPS II and contribute to defining MPS II-specific outcome measures for future clinical trials.

Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells.

The pathological consequences leading to primary storage, autophagy impairment, impaired mitochondrial dynamics, and endoplasmic reticulum (ER) stress on neural cell dysfunction and apoptosis in metachromatic leukodystrophy (MLD) have been poorly elucidated. In the present study, we generated 2 cell lines of patient-specific-induced pluripotent stem cells (iPSCs) and modeled the progression of pathological events during the differentiation of iPSCs to motor neuron progenitors (MNPs) and mature motor neurons (MNs). The iPS cells were generated from two late-infantile MLD patient-derived skin fibroblasts using electroporation or the Sendai virus. Olig2+ MNPs were generated from both iPSC lines using a combination of small molecules in a chemically defined neural medium. Furthermore, the MNPs could be differentiated into mature MNs, which was confirmed by RT-PCR and MN markers, including SMI32 and ChAT. The population of MNs was approximately 50% under the culture conditions. Pathological observation of MLD patient-derived iPSCs revealed lysosomal accumulation and impaired autophagy. In addition, both MNPs and MNs derived from MLD-iPSCs showed increased lysosomal accumulation, dysfunctional autophagy, impaired mitophagy, endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation, and premature cellular death.

A neuropathological cell model derived from Niemann-Pick disease type C patient-specific iPSCs shows disruption of the p62/SQSTM1-KEAP1-NRF2 Axis and impaired formation of neuronal networks.

Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by a recessive mutation in the NPC1 or NPC2 gene, in which patients exhibit lysosomal accumulation of unesterified cholesterol and glycolipids. Most of the research on NPC has been done in patient-derived skin fibroblasts or mouse models. Therefore, we developed NPC patient neurons derived from induced pluripotent stem cells (iPSCs) to investigate the neuropathological cause of the disease. Although an accumulation of cholesterol and glycolipids, which is characteristic of NPC, was observed in both undifferentiated iPSCs and derived neural stem cells (NSCs), we could not observed the abnormalities in differentiation potential and autophagic activity in such immature cells. However, definite neuropathological features were detected in mature neuronal cells generated from NPC patient-derived iPSCs. Abnormal accumulation of cholesterol and other lipids identified by lipid droplets and number of enlarged lysosomes was more prominent in mature neuronal cells rather than in iPSCs and/or NSCs. Thin-sectioning electron microscopic analysis also demonstrated numerous typical membranous cytoplasmic bodies in mature neuronal cells. Furthermore, TUJ1-positive neurite density was significantly reduced in NPC patient-derived neuronal cells. In addition, disruption of the p62/SQSTM1-KEAP1-NRF2 axis occurred in neurons differentiated from NPC patient-derived iPSCs. These data indicate the impairment of neuronal network formation associated with neurodegeneration in mature neuronal cells derived from patients with NPC.

A combination of 7-ketocholesterol, lysosphingomyelin and bile acid-408 to diagnose Niemann-Pick disease type C using LC-MS/MS.

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by mutations of NPC1 or NPC2, which encode the proteins that are responsible for intracellular cholesterol trafficking. Loss of this function results in the accumulation of cholesterol-related products, such as oxysterols, sphingolipids, and NPC-related bile acids, which were recently used as biochemical biomarkers for the diagnosis of NPC. Bile acid-408 is a new significant compound we found in Japanese NPC patients, and it likely belongs to the category of bile acids. However, the diagnosis of NPC using a single biomarker is not satisfactory for clinical application because of the high instance of false negatives or positives. Therefore, we proposed an application of NPC diagnosis using a combination of 7-ketocholesterol (7-KC), lysosphingomyelin (lysoSM), bile acid-408 and/or glucosylsphingosine (lysoGL-1). METHODS AND FINDINGS: 7-KC, lysoSM and lysoGL-1 in sera and bile acid-408 in dried blood spots (DBS) were quantified within 17 minutes using tandem mass spectrometry and high-resolution mass spectrometry, respectively. We measured these biomarkers in NPC patients (n = 19), X-linked adrenoleukodystrophy (X-ALD) patients (n = 5), patients with other lysosomal diseases (n = 300), newborns (n = 124) and healthy people (n = 74). Our results showed a promising accuracy (97%) for NPC diagnosis using the combination of 7-KC, lysoSM and bile acid-408. However, contrary to our expectations, lysoGL-1 levels did not present at a significantly greater amount in NPC patients than other patients and negative controls. CONCLUSIONS: The combination of 7-KC, lysoSM and bile acid-408 improves the accuracy of NPC diagnosis and is feasible for mass screening due to its simple sample preparation and measurement. Future research should investigate the chemical structure of bile acid-408 to further facilitate its advantage in diagnosis.

Future clinical and biochemical predictions of Fabry disease in females by methylation studies of the GLA gene.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A). The clinical variability of the phenotypes of Fabry disease in females is still poorly understood. The degree of aberrant methylation of non-mutated alleles is thought to have significant effects on X-chromosome inactivation (XCI). We previously reported that one heterozygous Fabry female showing classical phenotypes had complete methylation of the non-mutated allele of the GLA gene. In this report, we summarized 36 heterozygous females with a clinical severity score based on the FAbry STabilization indEX (FASTEX). We measured their α-gal A activity and plasma/ serum globotriaosylsphingosine (lyso-Gb3) accumulation and performed electron microscopy of skin biopsies. We analyzed the methylation-sensitive restriction enzyme sites throughout the GLA gene, including the 5'UTR, and found a single SacII site and multiple HhaI and HpaII sites aggregated in exon 1 and the 5'UTR. One HpaII sequence in exon 7 was also detected as a methylation-sensitive site. With methylation-sensitive restriction enzymes, methylated and non-methylated alleles could be separated, and the ratio of the methylation was quantified. We found a clear correlation between the severity of the phenotype and lyso-Gb3 accumulation for heterozygous Fabry disease in females. Methylation of the non-mutated allele was also proportionately correlated to the clinical severity score measured by FASTEX.

Dysregulated DNA methylation of GLA gene was associated with dysfunction of autophagy.

Lysosomes are an essential organ for cellular metabolism and play an important role in autophagy. We examined the association between methylation and autophagy in a severely affected female patient with Fabry disease, which is caused by mutation of the GLA gene on the X chromosome, and her two sisters, who had few symptoms. We confirmed autophagic flux by LC3 turnover assay using fibroblasts from each sister. In the severe female patient, autophagic flux showed abnormal while her two sisters with few symptoms had normal autophagic flux, revealing the direct relationship between symptoms and autophagic flux. Furthermore, we observed the levels of p62, which is a substrate for autophagy, and lysosome morphology. In the severe patient of this family, lysosomes were enlarged and p62 was accumulated. The methylated allele of the GLA gene in the severe patient had a high proportion of wild alleles; conversely, the sisters' methylated allele had a high proportion of mutant alleles. Therefore, we examined the mRNA expression level of the mutant allele by allele-specific PCR. It was high in the severe patient and low in the siblings with few symptoms. That is, the correlation between the mRNA expression level of the mutant allele and disease severity was confirmed. We showed a correlation between severe symptoms, dysfunction of autophagy and methylation of wild alleles in Fabry disease. It was suggested that allele-specific PCR may lead to a diagnosis and help to determine the prognosis of female patients with Fabry disease.

A Case of Adult-onset Pompe Disease with Cerebral Stroke and Left Ventricular Hypertrophy.

BACKGROUND: Pompe disease is an autosomal recessive glycogen storage disorder caused by a deficiency of the lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase. The adult-onset form, late-onset Pompe disease, has been characterized by glycogen accumulation, primarily in skeletal and smooth muscles, causing weakness of the proximal limb girdle and respiratory compromises. CASE REPORT: A 59-year-old female was admitted to the hospital with acute cerebral stroke at the age of 57years. Following her admission, conventional conservative stroke management followed by cerebral arterial clipping was performed. However, weakness of lower extremities, predominantly in the right side, and evening headache were persisting. After obtaining a careful past history, she noticed that she had a history of recurrent respiratory tract infection and she did not like any physical exercise in school. She also complained of gait disturbance since 32years of age. She had also been suffering from systemic hypertension since 40years of age. She had mild respiratory and swallowing difficulties. Her brain Magnetic Resonance (MR) revealed multiple infractions and white matter degeneration with irregular basilar arterial walls. A computed tomography (CT) scan of lower extremities showed diffuse fibrosis of the proximal muscles predominantly on the right thigh. Cardiac echocardiogram showed left ventricular hypertrophy. Electron microscopy of blood cells including lymphocytes and platelets and skin fibroblasts showed marked granular inclusions in lysosomes, suggesting glycogen accumulation. Her measured acid α-glucosidase activity was very low, 1.3 pmol hour-1 punch-1, and we found a homozygous splice-site mutation c.546G>T in the GAA gene. CONCLUSION: Cerebral stoke as an initial finding for an adult-type Pompe disease is rare. Left ventricular hypertrophy is also rarely reported for adult onset of Pompe disease. This case will explore further ways to diagnose adult-onset Pompe disease.

A Simple and Sensitive Liquid Chromatography with Tandem Mass Spectrometric Method for the Simultaneous Determination of Anthraquinone Glycosides and Their Aglycones in Rat Plasma: Application to a Pharmacokinetic Study of Rumex acetosa Extract.

Rumex acetosa (R. acetosa) has been used in folk remedies for gastrointestinal disorders and cutaneous diseases. Rumex species, in particular, contain abundant anthraquinones. Anthraquinone glycosides and aglycones show different bioactive effects. However, information on the pharmacokinetics of anthraquinone glycosides is limited, and methods to quantify anthraquinone glycosides in plasma are rarely available. A simple and sensitive liquid chromatography-tandem mass spectrometric bioanalytical method for the simultaneous determination of both anthraquinone glycosides and their aglycones, including emodin, emodin-8-O-ß-d-glucoside, chrysophanol, chrysophanol-8-O-ß-d-glucoside, physcion, and physcion-8-O-ß-d-glucoside , in a low volume of rat plasma (20 µL) was established. A simple and rapid sample preparation was employed using methanol as a precipitating agent with appropriate sensitivity. Chromatographic separation was performed on HPLC by using a biphenyl column with a gradient elution using 2 mM ammonium formate (pH 6) in water and 2 mM ammonium formate (pH 6) in methanol within a run time of 13 min. The anthraquinones were detected on triple-quadrupole mass spectrometer in negative ionization mode using multiple-reaction monitoring. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability. The values of the lower limit of quantitation of anthraquinones were 1⁻20 ng/mL. The intra-batch and inter-batch accuracies were 96.7⁻111.9% and the precision was within the acceptable limits. The method was applied to a pharmacokinetic study after oral administration of R. acetosa 70% ethanol extract to rats at a dose of 2 g/kg.

Unfolded protein response is activated in Krabbe disease in a manner dependent on the mutation type.

Krabbe disease, one of the autosomal-recessive lysosomal storage disorders (LSDs), is caused by a deficiency of galactocerebrosidase (GALC) activity, resulting in the intracellular accumulation of psychosine, which is cytotoxic for neuronal cells. Genetically pathogenic mutations result in conformational changes in GALC and disrupt the lysosmal trafficking of cargos, which subsequently accumulate in the endoplasmic reticulum (ER). Recently, ER stress together with the activation of the unfolded protein response (UPR) has been suggested to play a key role in the pathogenesis of LSDs. In this study, we hence investigated whether the UPR is activated in Krabbe disease using COS-7 cells expressing pathogenic GALC mutants and skin fibroblasts (SFs) from Krabbe disease patients with various phenotypes, using a combination of semiquantitative and quantitative real-time polymerase chain reactions. We found that UPR activation in Krabbe disease depends on the mutations and cell types, and there is the possibility that multiple pathways, involving ER chaperones, inositol-requiring kinase 1, and protein kinase regulated by RNA-like ER kinase are activated by mutations associated with the infantile form. These results indicate that in Krabbe disease, each misfolded/unfolded protein evokes different UPR activation depending on the mutation, and that the activated pathways affect the phenotypes.

Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening.

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS.

Ten-year-long enzyme replacement therapy shows a poor effect in alleviating giant leg ulcers in a male with Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A), leading to the progressive accumulation of glycosphingolipids. Classical hemizygous males usually present symptoms, including pain and paresthesia in the extremities, angiokeratoma, hypo- or anhidrosis, abdominal pain, cornea verticillata, early stroke, tinnitus, and/or hearing loss, during early childhood or adolescence. Moreover, proteinuria, renal impairment, and cardiac hypertrophy can appear with age. Enzyme replacement is the most common therapy for Fabry disease at present which has been approved in Japan since 2004. We report a case involving a 27-year-old male with extreme terminal pain, anhidrosis, abdominal pain, tinnitus, hearing impairment, cornea verticillata, and recurrent huge ulcers in the lower extremities. At the age of 16 years, he was diagnosed with Fabry disease with a positive family history and very low α-gal A activity. He then received enzyme replacement therapy (ERT) with recombinant human agalsidase beta at 1 mg/kg every 2 weeks for 10 years. Throughout the course of ERT, his leg ulcers recurred, and massive excretion of urinary globotriaosylceramide and plasma globotriaosylsphingosine was observed. Electron microscopy of the venous tissue in the regions of the ulcer showed massive typical zebra bodies in the vascular wall smooth muscle cells.

Application of a diagnostic methodology by quantification of 26:0 lysophosphatidylcholine in dried blood spots for Japanese newborn screening of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a rare inherited metabolic disease that results in the accumulation of very long chain fatty acids (VLCFA) in plasma and all tissues. Recent studies regarding cerebral X-ALD (CALD) treatment emphasize the importance of its early diagnosis. 26:0 lysophosphatidylcholine (LysoPC) is a sensitive biomarker for newborn screening of X-ALD, while its application for Japanese DBS is unclear. Therefore, we evaluated the feasibility of 20:0 LysoPC and 24:0 LysoPC along with 26:0 LysoPC for diagnosing X-ALD in a cohort of newborns (n = 604), healthy adults (n = 50) and patients (n = 4). Results indicated that 26:0 LysoPC had strong significance for discrimination of patients by the amounts of 2.0 to 4.0 and 0.1 to 1.9 pmol/punch for patients and newborns/healthy adults, respectively. Based on these values, we recommend that further diagnostic confirmation is essential if the amount of 26:0 LysoPC in DBS is above 1.7 pmol/punch.

Early onset of Fazio-Londe syndrome: the first case report from the Arabian Peninsula.

Fazio-Londe syndrome is a rare neurological disorder presenting with sensorineural deafness, bulbar palsy and respiratory compromise that is caused by mutation in the SLC52A3 gene, which encodes the intestinal (hRFT2) riboflavin transporter. We report a patient with early onset of Fazio-Londe syndrome as the first case report in Saudi Arabia with rapid regression to death at 24 months of age.

The severe clinical phenotype for a heterozygous Fabry female patient correlates to the methylation of non-mutated allele associated with chromosome 10q26 deletion syndrome.

Heterozygous Fabry females usually have an attenuated form of Fabry disease, causing them to be symptomatic; however, in rare cases, they can present with a severe phenotype. In this study, we report on a 37-year-old woman with acroparesthesia, a dysmorphic face, left ventricular hypertrophy, and intellectual disability. Her father had Fabry disease and died due to chronic renal and congestive cardiac failure. Her paternal uncle had chronic renal failure and intellectual disability, and her paternal aunt was affected with congestive cardiac failure. The patient has two sisters with no significant medical illness. However, her nephew has acroparesthesia, anhidrosis, and school phobia, and her niece shows mild phenotypes. The patient's enzyme analysis showed very low α-galactosidase A (α-gal A) activity in dried blood spot (DBS), lymphocytes, and skin fibroblasts with massive excretion of Gb3 and Gb2 in urine and lyso-Gb3 in DBS and plasma. Electron microscopic examination showed a large accumulation of sphingolipids in vascular endothelial cells and keratinocytes. Chromosomal analysis and comparative genomic hybridization microarray showed 10q26 terminal deletion. Molecular data showed a novel heterozygous stop codon mutation in exon 1 of the GLA gene in her sisters and niece, and a hemizygous state in her nephew. When we checked the methylation status, we found her non-mutated allele in the GLA gene was methylated. However, the non-mutated alleles of her sisters were non-methylated, and those of her niece were partially methylated. The chromosomal and methylation study may speculate the severity of her clinical phenotypes.

Thirteen year retrospective review of the spectrum of inborn errors of metabolism presenting in a tertiary center in Saudi Arabia.

BACKGROUND: Inborn errors of metabolism (IEMs) are individually rare; however, they are collectively common. More than 600 human diseases caused by inborn errors of metabolism are now recognized, and this number is constantly increasing as new concepts and techniques become available for identifying biochemical phenotypes. The aim of this study was to determine the type and distribution of IEMs in patients presenting to a tertiary care center in Saudi Arabia. METHOD: We conducted a retrospective review of children diagnosed with IEMs presenting to the Pediatric Department of King Abdulaziz Medical City in Riyadh, Saudi Arabia over a 13-year period. RESULTS: Over the 13- year period of this retrospective cohort, the total number of live births reached 110,601. A total of 187 patients were diagnosed with IEMs, representing a incidence of 169 in 100,000 births (1:591). Of these, 121 patients (64.7 %) were identified to have small molecule diseases and 66 (35.3 %) to have large molecule diseases. Organic acidemias were the most common small molecule IEMs, while lysosomal storage disorders (LSD) were the most common large molecule diseases. Sphingolipidosis were the most common LSD. CONCLUSION: Our study confirms the previous results of the high rate of IEMs in Saudi Arabia and urges the health care strategists in the country to devise a long-term strategic plan, including an IEM national registry and a high school carrier screening program, for the prevention of such disorders. In addition, we identified 43 novel mutations that were not described previously, which will help in the molecular diagnosis of these disorders.

Design and Implementation of a Novel Compatible Encoding Scheme in the Time Domain for Image Sensor Communication.

This paper presents a modulation scheme in the time domain based on On-Off-Keying and proposes various compatible supports for different types of image sensors. The content of this article is a sub-proposal to the IEEE 802.15.7r1 Task Group (TG7r1) aimed at Optical Wireless Communication (OWC) using an image sensor as the receiver. The compatibility support is indispensable for Image Sensor Communications (ISC) because the rolling shutter image sensors currently available have different frame rates, shutter speeds, sampling rates, and resolutions. However, focusing on unidirectional communications (i.e., data broadcasting, beacons), an asynchronous communication prototype is also discussed in the paper. Due to the physical limitations associated with typical image sensors (including low and varying frame rates, long exposures, and low shutter speeds), the link speed performance is critically considered. Based on the practical measurement of camera response to modulated light, an operating frequency range is suggested along with the similar system architecture, decoding procedure, and algorithms. A significant feature of our novel data frame structure is that it can support both typical frame rate cameras (in the oversampling mode) as well as very low frame rate cameras (in the error detection mode for a camera whose frame rate is lower than the transmission packet rate). A high frame rate camera, i.e., no less than 20 fps, is supported in an oversampling mode in which a majority voting scheme for decoding data is applied. A low frame rate camera, i.e., when the frame rate drops to less than 20 fps at some certain time, is supported by an error detection mode in which any missing data sub-packet is detected in decoding and later corrected by external code. Numerical results and valuable analysis are also included to indicate the capability of the proposed schemes.

Chemical chaperone treatment for galactosialidosis: Effect of NOEV on ß-galactosidase activities in fibroblasts.

INTRODUCTION: Galactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of GM1 ß-galactosidase (ß-gal) and neuraminidase secondary to a defect of a lysosomal enzyme protective protein/cathepsin A (PPCA) and mutation in CTSA gene. Three subtypes are recognized: early infantile, late infantile, and juvenile/adult. There is no specific therapy for patients with galactosialidosis at this time. OBJECTIVES: The aim of this study was to determine the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on ß-gal proteins in skin fibroblasts of PPCA-deficit patients. METHODS: ß-Gal and neuraminidase activities were measured for the diagnosis of the patients with galactosialidosis. Western blotting for PPCA protein and direct sequencing for CTSA gene were performed. Cultured skin fibroblast were treated with NOEV. RESULTS: We report four novel patients with galactosialidosis: one had the early infantile form and the other three had the juvenile/adult form. We found that NOEV stabilized ß-gal activity in lysate from cultured skin fibroblasts from these patients. Treatment with NOEV significantly enhanced ß-gal activity in cultured skin fibroblasts in the absence of PPCA. CONCLUSIONS: Our results indicate the possibility that NOEV chaperone therapy might have a beneficial effect, at least in part, for patients with galactosialidosis.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.

Late-onset Krabbe disease is predominant in Japan and its mutant precursor protein undergoes more effective processing than the infantile-onset form.

Krabbe disease is an autosomal recessive leukodystrophy caused by the deficiency of the galactocerebrosidase (GALC) enzyme. It is pathologically characterized by demyelination of the central and peripheral nervous systems by accumulation of galactosylsphingosine. To date, more than 120 mutations in the GALC gene have been reported worldwide and genotype-phenotype correlations have been reported in some types of mutations. In this study, we analyzed 22 unreported Japanese patients with Krabbe disease and summarized a total of 51 Japanese patients, including 29 previously reported patients. To elucidate how GALC mutations impair enzymatic activity, multiple disease-causing mutations including common mutations and polymorphisms were investigated for enzymatic activity and precursor processing ability with transient expression system. We also performed 3-D enzyme structure analysis to determine the effect of each new mutation. Five novel mutations were detected including one deletion c.1808delT [p.L603X], one nonsense mutation c.1023C>G [p.Y341X], and three missense mutations c.209T>C [p.L70P], c.1054G>A [p.G352R], and c.1937G>C [p.G646A]. For the total of 51 patients, 59% had late-onset forms of Krabbe disease. Seven common mutations accounted for 58% of mutant alleles of patients with Krabbe disease in Japan. Infantile-onset mutations had almost no enzyme activity, while late-onset mutations had 4%-20% of normal enzyme activity. The processing rate of precursor GALC protein to mature form was slower for infantile-onset mutations. Heat stability of the mutant proteins revealed that p.G270D was more stable compared to the other mutations. The constructed 3D-model showed that the residues for Krabbe mutations were less solvent-accessible and located in the core region of GALC protein. In conclusion, we have demonstrated that the most common phenotype in Japan is the late-onset type, that the enzyme activity for GALC mutants is correlated with mutational severity, and that the most pathogenic factor is due to the processing rate from the precursor to the mature protein.

A novel homozygous GALC mutation: very early onset and rapidly progressive Krabbe disease.

A clear cut genotype-phenotype correlation for Krabbe disease is not available. Therefore, it is important to identify new mutations and their associated phenotypes to predict the prognosis of the disease. The aim of this study is to identify the causative mutation(s) in a family with Krabbe disease. After a clinical evaluation and suspicion of Krabbe disease galactocerebrosidase activity was analyzed and GALC gene mutation analysis was performed. The galactocerebrosidase enzyme activity was 0.01 nmol/mg/h protein (normal range 0.8-4). For further investigation mutation screening was performed by Sanger sequencing across the 17 exons of GALC gene. A novel homozygous mutation c.727delT (p.S243QfsX7) was found. In this study we present the clinical findings along with a novel GALC mutation in a consanguineous Turkish family. Although the relationship between the various genotypes and phenotypes in Krabbe disease has not been fully elucidated an accurate genetic family study is helpful for genetic counseling follow-up and therapy of Krabbe disease. Also, it is important to identify new mutations in order to clarify their clinical importance, to assess the prognosis of the disease, and to suggest either prenatal diagnosis or preimplantation genetic diagnosis to the effected families.