Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Lentivirus-modified autologous hematopoietic stem cell gene therapy (HSCGT) has recently been approved for clinical use in pre and early symptomatic children with MLD to increase ARSA activity. Unfortunately, this advanced therapy is not available for most patients with MLD who have progressed to more advanced symptomatic stages at diagnosis. Patients with late-onset juvenile MLD typically present with a slower neurological progression of symptoms and represent a significant burden to the economy and healthcare system, whereas those with early onset infantile MLD die within a few years of symptom onset. We conducted a pilot study to determine the safety and benefit of HSCGT in patients with postsymptomatic juvenile MLD and report preliminary results. The safety profile of HSCGT was favorable in this long-term follow-up over 9 years. The most common adverse events (AEs) within 2 months of HSCGT were related to busulfan conditioning, and all AEs resolved. No HSCGT-related AEs and no evidence of distorted hematopoietic differentiation during long-term follow-up for up to 9.6 years. Importantly, to date, patients have maintained remarkably improved ARSA activity with a stable disease state, including increased Functional Independence Measure (FIM) score and decreased magnetic resonance imaging (MRI) lesion score. This long-term follow-up pilot study suggests that HSCGT is safe and provides clinical benefit to patients with postsymptomatic juvenile MLD.
Humans ; Leukodystrophy, Metachromatic/genetics* ; Pilot Projects ; Genetic Therapy/methods* ; Hematopoietic Stem Cell Transplantation ; Male ; Follow-Up Studies ; Female ; Lentivirus/genetics* ; Child ; Child, Preschool ; Hematopoietic Stem Cells/metabolism* ; Cerebroside-Sulfatase/metabolism* ; Adolescent

Objective: aaWilson’s disease (WD) is a rare genetic disorder of copper metabolism, and longitudinal follow-up studies are limited. We performed a retrospective analysis to determine the clinical characteristics and long-term outcomes in a large WD cohort. Methods: aaMedical records of WD patients diagnosed from 2006–2021 at National Taiwan University Hospital were retrospectively evaluated for clinical presentations, neuroimages, genetic information, and follow-up outcomes. Results: aaThe present study enrolled 123 WD patients (mean follow-up: 11.12 ± 7.41 years), including 74 patients (60.2%) with hepatic features and 49 patients (39.8%) with predominantly neuropsychiatric symptoms. Compared to the hepatic group, the neuropsychiatric group exhibited more Kayser-Fleischer rings (77.6% vs. 41.9%, p < 0.01), lower serum ceruloplasmin levels (4.9 ± 3.9 vs. 6.3 ± 3.9 mg/dL, p < 0.01), smaller total brain and subcortical gray matter volumes (p < 0.0001), and worse functional outcomes during follow-up (p = 0.0003). Among patients with available DNA samples (n = 59), the most common mutations were p.R778L (allelic frequency of 22.03%) followed by p.P992L (11.86%) and p.T935M (9.32%). Patients with at least one allele of p.R778L had a younger onset age (p = 0.04), lower ceruloplasmin levels (p < 0.01), lower serum copper levels (p = 0.03), higher percentage of the hepatic form (p = 0.03), and a better functional outcome during follow-up (p = 0.0012) compared to patients with other genetic variations. Conclusion aaThe distinct clinical characteristics and long-term outcomes of patients in our cohort support the ethnic differences regarding the mutational spectrum and clinical presentations in WD.

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset alpha-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75+/-0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77+/-0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.
Adolescent ; Adult ; Blood Chemical Analysis/*methods ; Child ; Chromatography, High Pressure Liquid ; *Dried Blood Spot Testing ; Fabry Disease/blood/diagnosis ; Female ; Glycolipids/*blood ; Humans ; Infant, Newborn ; Male ; Sphingolipids/*blood ; Tandem Mass Spectrometry ; Young Adult

OBJECTIVENeonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, OMIM #605814) is a novel autosomal recessive disease caused by mutations in the gene SLC25A13 that encodes for citrin, a liver-type aspartate/glutamate carrier located in the mitochondrial inner membrane. SLC25A13 was cloned in 1999 by Kobayashi et al at Kagoshima University in Japan, and until now, most of the NICCD patients reported in the world were Japanese. Most of the Chinese NICCD patients diagnosed by genetic analysis had the same SLC25A13 mutations as Japanese, however, in some cases, known mutations were not detected. This research aimed to identify novel SLC25A13 mutations in Chinese NICCD patients and to explore the experimental conditions for their genetic diagnosis.

METHODSGenomic DNA was extracted from blood samples of 3 NICCD patients from Taiwan (P757), Guangdong (P1194) and Hebei province (P1443) of China, respectively, and all the 18 exons and their flanking sequences of SLC25A13 gene were sequenced. Furthermore, the identified novel mutations were diagnosed by amplification with PCR, digestion with corresponding restriction endonuclease, and agarose gel electrophoresis.

RESULTSThree novel mutations identified in SLC25A13 gene of the 3 NICCD patients were an abnormal splicing IVS7-2A > G (P757), a missense A541D (c.1622C > A, P1194) and a nonsense R319X (c.955C > T, P1443). The PCR-restriction fragment length polymorphism (RFLP) procedures for their genetic diagnosis were also established, with specific fragments on electrophoresis after digestion of the PCR products with three different restriction endonucleases Msp I, Hpy188I and Taq I, respectively.

CONCLUSIONSSo far as we know, the three novel mutations in SLC25A13 gene of Chinese NICCD patients were first identified, suggesting that SLC25A13 mutation distributed in Chinese population is somewhat different from that in Japanese. Moreover, the PCR-RFLP diagnostic procedures established in this research provide valuable tools not only for the genetic diagnosis of NICCD but also for further molecular epidemiologic investigations in Chinese population.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Calcium-Binding Proteins ; deficiency ; Child, Preschool ; Cholestasis, Intrahepatic ; diagnosis ; etiology ; genetics ; Female ; Humans ; Infant ; Male ; Mitochondrial Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Organic Anion Transporters ; deficiency

Objective Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, OMIM 605814 ) is a novel autosomal recessive disease results from mutations in the gene SLC25A13 that encodes for citrin, a liver-type aspartate/glutamate cartier located in the mitochondrial inner membrane. Most of the Chinese NICCD patients diagnosed by genetic analysis had the sameSLC25A13 mutations as Japanese, however, in some cases, the known mutations were not detected. This research aimed to identify novel SLC25A13 mutations in Chinese NICCD patients and to explore the experimental conditions for their genetic diagnosis.Methods Genomic DNA was extracted from blood samples of 3 NICCD patients from Taiwan (P757), Guangdong (P1194) and Hebei (P1443) Province of China, respectively; and all the 18 exons and their flanking sequences of SLC25A13 gene were sequenced. Furthermore, the identified novel mutations were diagnosed by amplification with PCR, digestion with corresponding restriction endonuclease, and agarose gel electrophoresis.Results Three novel mutations identified in SLC25A13 gene of the 3 NICCD patients were an abnormal splicing IVS7-2A>G (P757), a missense A541D (c. 1622C > A, P1194) and a nonsense R319X (c. 955C > T, P1443). The PCR-RFLP procedures for their genetic diagnosis were also established, with specific fragments on electrophoresis after digestion of the PCR products with three different restriction endonucleases Msp Ⅰ, Hpy188Ⅰ and Taq Ⅰ, respectively.Conclusions The three novel mutations in SLC25A13 gene of Chinese NICCD patients were first identified, suggesting that SLC25A13 mutation distributed in Chinese population is somewhat different from that in Japanese. Moreover, the PCR-RFLP diagnostic procedures established in this research provide valuable tools not only for the genetic diagnosis of NICCD but also for further molecular epidemiologic investigations in Chinese population.Acknowledgement We are grateful to all research subjects and their family members for their cooperation, and to many members of medical staff who contributed much to this research. This study was financially supported partially by Guangdong Provincial Research Grant for Science and Technology (No. 2004B50301008) and the Major State Basic Research Development Program of China (No. 2007CB511901 ), and by Grants-in-Aid for Scientific Research (B: Nos. 16390100 & 19390096 ) and for Asia-Africa Scientific Platform Program (AASPP) from the Japan Society for the Promotion of Science.