Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants.

PURPOSE: To characterize the molecular genetics of autosomal recessive Noonan syndrome. METHODS: Families underwent phenotyping for features of Noonan syndrome in children and their parents. Two multiplex families underwent linkage analysis. Exome, genome, or multigene panel sequencing was used to identify variants. The molecular consequences of observed splice variants were evaluated by reverse-transcription polymerase chain reaction. RESULTS: Twelve families with a total of 23 affected children with features of Noonan syndrome were evaluated. The phenotypic range included mildly affected patients, but it was lethal in some, with cardiac disease and leukemia. All of the parents were unaffected. Linkage analysis using a recessive model supported a candidate region in chromosome 22q11, which includes LZTR1, previously shown to harbor mutations in patients with Noonan syndrome inherited in a dominant pattern. Sequencing analyses of 21 live-born patients and a stillbirth identified biallelic pathogenic variants in LZTR1, including putative loss-of-function, missense, and canonical and noncanonical splicing variants in the affected children, with heterozygous, clinically unaffected parents and heterozygous or normal genotypes in unaffected siblings. CONCLUSION: These clinical and genetic data confirm the existence of a form of Noonan syndrome that is inherited in an autosomal recessive pattern and identify biallelic mutations in LZTR1.

Hematopoietic Stem Cell Transplantation for Patients with Mucopolysaccharidosis II.

There is limited information regarding the long-term outcomes of hematopoietic stem cell transplantation (HSCT) for mucopolysaccharidosis II (MPS II). In this study, clinical, biochemical, and radiologic findings were assessed in patients who underwent HSCT and/or enzyme replacement therapy (ERT). Demographic data for 146 HSCT patients were collected from 27 new cases and 119 published cases and were compared with 51 ERT and 15 untreated cases. Glycosaminoglycan (GAG) levels were analyzed by liquid chromatography tandem mass spectrometry in blood samples from HSCT, ERT, and untreated patients as well as age-matched controls. Long-term magnetic resonance imaging (MRI) findings were investigated in 13 treated patients (6 ERT and 7 HSCT). Mean age at HSCT was 5.5 years (range, 2 to 21.4 years) in new patients and 5.5 years (range, 10 months to 19.8 years) in published cases. None of the 27 new patients died as a direct result of the HSCT procedure. Graft-versus-host disease occurred in 8 (9%) out of 85 published cases, and 9 (8%) patients died from transplantation-associated complications. Most HSCT patients showed greater improvement in somatic features, joint movements, and activity of daily living than the ERT patients. GAG levels in blood were significantly reduced by ERT and levels were even lower after HSCT. HSCT patients showed either improvement or no progression of abnormal findings in brain MRI while abnormal findings became more extensive after ERT. HSCT seems to be more effective than ERT for MPS II in a wide range of disease manifestations and could be considered as a treatment option for this condition.

Glycosaminoglycan levels in dried blood spots of patients with mucopolysaccharidoses and mucolipidoses.

Mucopolysaccharidoses (MPSs) and mucolipidoses (ML) are groups of lysosomal storage disorders in which lysosomal hydrolases are deficient leading to accumulation of undegraded glycosaminoglycans (GAGs), throughout the body, subsequently resulting in progressive damage to multiple tissues and organs. Assays using tandem mass spectrometry (MS/MS) have been established to measure GAGs in serum or plasma from MPS and ML patients, but few studies were performed to determine whether these assays are sufficiently robust to measure GAG levels in dried blood spots (DBS) of patients with MPS and ML. MATERIAL AND METHODS: In this study, we evaluated GAG levels in DBS samples from 124 MPS and ML patients (MPS I=16; MPS II=21; MPS III=40; MPS IV=32; MPS VI=10; MPS VII=1; ML=4), and compared them with 115 age-matched controls. Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Subsequently, dermatan sulfate (DS), heparan sulfate (HS-0S, HS-NS), and keratan sulfate (mono-sulfated KS, di-sulfated KS, and ratio of di-sulfated KS in total KS) were measured by MS/MS. RESULTS: Untreated patients with MPS I, II, VI, and ML had higher levels of DS compared to control samples. Untreated patients with MPS I, II, III, VI, and ML had higher levels of HS-0S; and untreated patients with MPS II, III and VI and ML had higher levels of HS-NS. Levels of KS were age dependent, so although levels of both mono-sulfated KS and di-sulfated KS were generally higher in patients, particularly for MPS II and MPS IV, age group numbers were not sufficient to determine significance of such changes. However, the ratio of di-sulfated KS in total KS was significantly higher in all MPS patients younger than 5years old, compared to age-matched controls. MPS I and VI patients treated with HSCT had normal levels of DS, and MPS I, VI, and VII treated with ERT or HSCT had normal levels of HS-0S and HS-NS, indicating that both treatments are effective in decreasing blood GAG levels. CONCLUSION: Measurement of GAG levels in DBS is useful for diagnosis and potentially for monitoring the therapeutic efficacy in MPS.

The clinical features of osteogenesis imperfecta in Vietnam.

PURPOSE: Osteogenesis imperfecta (OI) has not been studied in a Vietnamese population before. The aim of this study was to systematically collect epidemiological information, investigate clinical features and create a clinical database of OI patients in Vietnam for future research and treatment strategy development. METHOD: Participants underwent clinical and physical examinations; also medical records were reviewed. Genealogical information was collected and family members' phenotypical manifestations recorded. Cases were classified according to the Sillence classification. RESULTS: In total, 146 OI patients from 120 families were studied: 46 with OI Type I, 46 with Type III and 54 with Type IV. Almost patients had skeletal deformations. One hundred and forty-two had a history of fractures, 117 blue sclera, 89 dentinogenesis imperfecta and 26 hearing loss. The total number of fractures was 1,932. Thirty-four patients had intra-uterine fractures and nine had perinatal fractures. Surgery was performed 163 times in 58 patients; 100 osteosyntheses and 63 osteotomies. Bisphosphonate treatment was used in 37 patients. The number of affected individuals and predominance of severe forms of OI indicate that the disease is under diagnosed in Vietnam, especially in cases without a family history or with mild form of OI. Deformities appeared in all patients with different severity and localisation, affecting mostly the lower limbs. OI medical and surgical treatment rates are low and in most cases surgery was performed due to fractures. CONCLUSIONS: Compared to previous studies, our results indicate a lower OI prevalence and greater severity of symptoms in the Vietnamese population when compared with other areas. Further investigation, improved diagnosis and treatment are needed to increase the patients' quality of life.

Enzyme replacement therapy in newborn mucopolysaccharidosis IVA mice: early treatment rescues bone lesions?

We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology [1]. To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in the liver and spleen, with detectable activity in the bone and brain. Second, newborn ERT was conducted after a tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th weeks were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in the liver and spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than that in adult mice treated with ERT; however, hyaline and fibrous cartilage cells in the femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mice.

Therapies for the bone in mucopolysaccharidoses.

Patients with mucopolysaccharidoses (MPS) have accumulation of glycosaminoglycans in multiple tissues which may cause coarse facial features, mental retardation, recurrent ear and nose infections, inguinal and umbilical hernias, hepatosplenomegaly, and skeletal deformities. Clinical features related to bone lesions may include marked short stature, cervical stenosis, pectus carinatum, small lungs, joint rigidity (but laxity for MPS IV), kyphoscoliosis, lumbar gibbus, and genu valgum. Patients with MPS are often wheelchair-bound and physical handicaps increase with age as a result of progressive skeletal dysplasia, abnormal joint mobility, and osteoarthritis, leading to 1) stenosis of the upper cervical region, 2) restrictive small lung, 3) hip dysplasia, 4) restriction of joint movement, and 5) surgical complications. Patients often need multiple orthopedic procedures including cervical decompression and fusion, carpal tunnel release, hip reconstruction and replacement, and femoral or tibial osteotomy through their lifetime. Current measures to intervene in bone disease progression are not perfect and palliative, and improved therapies are urgently required. Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and gene therapy are available or in development for some types of MPS. Delivery of sufficient enzyme to bone, especially avascular cartilage, to prevent or ameliorate the devastating skeletal dysplasias remains an unmet challenge. The use of an anti-inflammatory drug is also under clinical study. Therapies should start at a very early stage prior to irreversible bone lesion, and damage since the severity of skeletal dysplasia is associated with level of activity during daily life. This review illustrates a current overview of therapies and their impact for bone lesions in MPS including ERT, HSCT, gene therapy, and anti-inflammatory drugs.

Mucopolysaccharidosis IVA: correlation between genotype, phenotype and keratan sulfate levels.

Mucopolysaccharidosis IVA (MPS IVA) is caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to systemic skeletal dysplasia because of excessive storage of keratan sulfate (KS) in chondrocytes. In an effort to determine a precise prognosis and personalized treatment, we aim to characterize clinical, biochemical, and molecular findings in MPS IVA patients, and to seek correlations between genotype, phenotype, and blood and urine KS levels. Mutation screening of GALNS gene was performed in 55 MPS IVA patients (severe: 36, attenuated: 13, undefined: 6) by genomic PCR followed by direct sequence analysis. Plasma and urine KS levels were measured by ELISA method. Genotype/phenotype/KS correlations were assessed when data were available. Fifty-three different mutations including 19 novel ones (41 missense, 2 nonsense, 4 small deletions, 1 insertion, and 5 splice-site) were identified in 55 patients and accounted for 93.6% of the analyzed mutant alleles. Thirty-nine mutations were associated with a severe phenotype and ten mutations with an attenuated one. Blood and urine KS concentrations in MPS IVA patients were age-dependent and markedly higher than those in age-matched normal controls. Plasma and urine KS levels in MPS IVA patients with the severe phenotype were higher than in those with an attenuated form. This study provides evidence for extensive allelic heterogeneity of MPS IVA. Accumulation of mutations as well as clinical descriptions and KS levels allows us to predict clinical severity more precisely and should be used for evaluation of responses to potential treatment options.

Human Variome Project country nodes: documenting genetic information within a country.

The Human Variome Project (http://www.humanvariomeproject.org) is an international effort aiming to systematically collect and share information on all human genetic variation. The two main pillars of this effort are gene/disease-specific databases and a network of Human Variome Project Country Nodes. The latter are nationwide efforts to document the genomic variation reported within a specific population. The development and successful operation of the Human Variome Project Country Nodes are of utmost importance to the success of Human Variome Project's aims and goals because they not only allow the genetic burden of disease to be quantified in different countries, but also provide diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research, respectively. Here, we report the discussions and recommendations that resulted from the inaugural meeting of the International Confederation of Countries Advisory Council, held on 12th December 2011, during the 2011 Human Variome Project Beijing Meeting. We discuss the steps necessary to maximize the impact of the Country Node effort for developing regional and country-specific clinical genetics resources and summarize a few well-coordinated genetic data collection initiatives that would serve as paradigms for similar projects.

Genetic Etiology of Neonatal Diabetes Mellitus in Vietnamese Infants and Characteristics of Those With INS Gene Mutations.

Background: Neonatal diabetes mellitus (NDM) is a rare (1:90,000 newborns) but potentially devastating metabolic disorder characterized by hyperglycemia combined with low levels of insulin. Dominantly-acting insulin (INS) gene mutations cause permanent NDM through single amino acid changes in the protein sequence leading to protein misfolding, which is retained within the endoplasmic reticulum (ER), causing ER stress and ß-cell apoptosis. Over 90 dominantly-acting INS gene mutations have been identified in individuals with permanent NDM. Patients and Methods: The study included 70 infants diagnosed with NDM in the first year of life between May 2008 and May 2021 at the Vietnam National Children's Hospital. Sequencing analysis of all the genes known to cause NDM was performed at the Exeter Genomic Laboratory, UK. Clinical characteristics, molecular genetics, and annual data relating to glycemic control (HbA1c) and severe hypoglycemia of those with INS mutations were collected. The main outcomes of interest were HbA1c, daily insulin dose, growth, and cognitive/motor development. Results: Fifty-five of 70 infants (78.5%) with NDM harbored a mutation in a known disease-causing gene and of these, 10 had six different de novo heterozygous INS mutations. Mean gestational age was 38.1 ± 2.5 weeks and mean birth weight was 2.8 ± 0.5 g. They presented with NDM at 20 ± 17 weeks of age; 6/10 had diabetic ketoacidosis with pH 7.13 ± 0.26; plasma glucose level 32.6 ± 14.3 mmol/l and HbA1C 81 ± 15% mmol/mol. After 5.5 ± 4.8 years of insulin treatment, 9/10 have normal development with a developmental quotient of 80-100% and HbA1C 64 ± 7.3 mmol/mol, 9/10 have normal height, weight, and BMI on follow-up. Conclusions: We report a series of Vietnamese NDM cases with dominant INS mutations. INS mutations are the third commonest cause of permanent NDM. We recommend screening of the INS gene in all children diagnosed with diabetes in the first year of life.

Cockayne syndrome without UV-sensitivity in Vietnamese siblings with novel ERCC8 variants.

Cockayne syndrome (CS) is a rare progeroid disorder characterized by growth failure, microcephaly, photosensitivity, and premature aging, mainly arising from biallelic ERCC8 (CS-A) or ERCC6 (CS-B) variants. In this study we describe siblings suffering from classical Cockayne syndrome but without photosensitivity, which delayed a clinical diagnosis for 16 years. By whole-exome sequencing we identified the two novel compound heterozygous ERCC8 variants c.370_371del (p.L124Efs*15) and c.484G>C (p.G162R). The causality of the ERCC8 variants, of which one results in a frameshift and the other affects the WD3 domain, was tested and confirmed by a rescue experiment investigating DNA repair in H2O2 treated patient fibroblasts. Structural modeling of the p.G162R variant indicates effects on protein-protein interaction. This case shows the importance to test for ERCC6 and ERCC8 variants even if patients do not present with a complete CS phenotype.

Closing the Type 1 Diabetes gap in South-East Asia through government partnership working with non-government organisations.

This will be the first publication of Type 1 diabetes(T1D) outcomes in five low-middle-income countries (LMICs)-Laos, Malaysia, Vietnam, Cambodia and Myanmar in the Southeast Asia (SEA) region. The information obtained has been possible due to partnership programmes of non-government organisationAction4Diabetes (A4D) with defined local hospitalsthrough a Memorandum of Understandingsigned with the governments in SEAthat guarantees ongoing supplies of free insulin, blood glucose meter supplies, HbA1c tests and hospital emergency funds. PARTICIPANTS: Between 2020 and 2021, 383 children and young people with T1D who were active in the A4D supported programmes were reviewed including information on health coverage, multidisciplinary team management, diabetic ketoacidosis (DKA) on admission and insulin regimen. RESULTS: Mean HbA1c between 2020 and 2021 for patients in these LMICs arereported for the first time. The average glycaemic index in the five SEAcountries reviewed between 2020 and 2021 were high at 83 mmol/mol (9.7%). CONCLUSIONS: Government partnership working with non-government organisationsto support T1D from diagnosis to adulthood are the first steps to closing thegaps in many LMICs. Further epidemiological studies are needed to identify the glycaemic outcomes and DKA rates on admission for many of these countries.

Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

Molecular Genetics, Clinical Characteristics, and Treatment Outcomes of KATP-Channel Neonatal Diabetes Mellitus in Vietnam National Children's Hospital.

Background: Neonatal diabetes mellitus (NDM) is defined as insulin-requiring persistent hyperglycemia occurring within the first 6 months of life, which can result from mutations in at least 25 different genes. Activating heterozygous mutations in genes encoding either of the subunits of the ATP-sensitive K+ channel (KATP channel; KCNJ11 or ABCC8) of the pancreatic beta cell are the most common cause of permanent NDM and the second most common cause of transient NDM. Patients with NDM caused by KATP channel mutations are sensitive to sulfonylurea (SU) treatment; therefore, their clinical management can be improved by replacing insulin with oral agents. Patients and Methods: Seventy patients were diagnosed with NDM between May 2008 and May 2021 at Vietnam National Children's Hospital, and molecular genetic testing for all genes known to cause NDM was performed at the Exeter Genomic Laboratory, UK. Patients with ABCC8 or KCNJ11 mutations were transferred from insulin to oral SU. Clinical characteristics, molecular genetics, and annual data relating to glycemic control, SU dose, severe hypoglycemia, and side effects were collected. The main outcomes of interest were SU dose, SU failure (defined as permanent reintroduction of daily insulin), and glycemic control (HbA1c). Results: Fifty-four of 70 patients (77%) with NDM harbored a genetic mutation and of these; 27 (50%) had activating heterozygous mutations in ABCC8 or KCNJ11. A total of 21 pathogenic mutations were identified in the 27 patients, including 13 mutations in ABCC8 and 8 mutations in KCNJ11. Overall, 51% had low birth weight (below 3rd percentile), 23 (85%) were diagnosed before 3 months of age, and 23 (85%) presented with diabetic ketoacidosis. At diagnosis, clinical and biochemical findings (mean ± SD) were pH 7.16 ± 0.16; HCO3- , 7.9 ± 7.4 mmol/L; BE, -17.9 ± 9.1 mmol/L; HbA1C, 7.98% ± 2.93%; blood glucose, 36.2 ± 12.3 mmol/L; and C-peptide median, 0.09 (range, 0-1.61 nmol/l). Twenty-six patients were successfully transferred from insulin to SU therapy. In the remaining case, remission of diabetes occurred prior to transfer. Glycemic control on SU treatment was better than on insulin treatment: HbA1c and blood glucose level decreased from 7.58% ± 4.63% and 19.04 ± 14.09 mmol/L when treated with insulin to 5.8 ± 0.94% and 6.87 ± 3.46 mmol/L when treated with SU, respectively. Conclusions: This is the first case series of NDM patients with ABCC8/KCNJ11 mutations reported in Vietnam. SU is safe in the short term for these patients and more effective than insulin therapy, consistent with all studies to date. This is relevant for populations where access to and cost of insulin are problematic, reinforcing the importance of genetic testing for NDM.

Enzyme replacement therapy in a murine model of Morquio A syndrome.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to accumulation of keratan sulfate (KS) and chrondroitin-6-sulfate. The pharmacokinetics and biodistributions were determined for two recombinant human GALNSs produced in CHO cell lines: native GALNS and sulfatase-modifier-factor 1 (SUMF1) modified GALNS. Preclinical studies of enzyme replacement therapy (ERT) by using two GALNS enzymes were performed on MPS IVA mice. The half-lives in blood circulation of two phosphorylated GALNS enzymes were similar (native, 2.4 min; SUMF1, 3.3 min). After intravenous doses of 250 units/g body weight were administered, each enzyme was primarily recovered in liver and spleen, with detectable activity in other tissues including bone and bone marrow. At 4 h post-injection, enzyme activity was retained in the liver, spleen, bone and bone marrow at levels that were 20-850% of enzyme activity in the wild-type mice. After intravenous doses of 250 units/g of native GALNS, and 250, 600 or 1000 units/g of SUMF1-GALNS were administered weekly for 12 weeks, MPS IVA mice showed marked reduction of storage in visceral organs, sinus lining cells in bone marrow, heart valves, ligaments and connective tissues. A dose-dependent clearance of storage material was observed in brain. The blood KS level assayed by tandem mass spectrometry was reduced nearly to normal level. These preclinical studies demonstrate the clearance of tissue and blood KS by administered GALNS, providing the in vivo rationale for the design of ERT trials in MPS IVA.

Validation of disaccharide compositions derived from dermatan sulfate and heparan sulfate in mucopolysaccharidoses and mucolipidoses II and III by tandem mass spectrometry.

Glycosaminoglycans (GAGs) are accumulated in various organs in both mucopolysaccharidoses (MPS) and mucolipidoses II and III (ML II and III). MPS and ML II and III patients can not properly degrade dermatan sulfate (DS) and/or heparan sulfate (HS). HS storage occurs in the brain leading to neurological signs while DS storage involves mainly visceral and skeletal manifestations. Excessive DS and HS released into circulation and thus blood levels of both are elevated, therefore, DS and HS in blood could be critical biomarkers for MPS and ML. Such measurement can provide a potential early screening, assessment of the clinical course and efficacy of therapies. We here assay DS and HS levels in MPS and ML patients using liquid chromatography tandem mass spectrometry (LC/MS/MS). Plasma samples were digested by heparitinase and chondroitinase B to obtain disaccharides of DS and HS, followed by LC/MS/MS analysis. One hundred-twenty samples from patients and 112 control samples were analyzed. We found that all MPS I, II, III and VI patients had a significant elevation of all DS+HS compositions analyzed in plasma, compared with the controls (P<0.0001). Specificity and sensitivity was 100% if the cut off value is 800 ng/ml between control and these types of MPS group. All MPS I, II and III patients also had a significant elevation of plasma HS, compared with the controls (P<0.0001). All MPS VI patients had a significant elevation of plasma DS, compared with the controls (P<0.0001). These findings suggest measurement of DS and/or HS levels by LC/MS/MS is applicable to the screening for MPS I, II, III and VI patients.

Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography-tandem mass spectrometry.

Mucopolysaccharidosis type IVA (MPS IVA, Morquio A disease), a progressive lysosomal storage disease, causes skeletal chondrodysplasia through excessive storage of keratan sulfate (KS). KS is synthesized mainly in cartilage and released to the circulation. The excess storage of KS disrupts cartilage, consequently releasing more KS into circulation, which is a critical biomarker for MPS IVA. Thus, assessment of KS level provides a potential screening strategy and determines clinical course and efficacy of therapies. We have recently developed a tandem mass spectrometry liquid chromatography [LC/MS/MS] method to assay KS levels in blood. Forty-nine blood specimens from patients with MPS IVA [severe (n = 33), attenuated (n = 11) and undefined (n = 5)] were analyzed for comparison of blood KS concentration with that of healthy subjects and for correlation with clinical severity. Plasma samples were digested by keratanase II to obtain disaccharides of KS. Digested samples were assayed by LC/MS/MS. We found that blood KS levels (0.4-26 µg/ml) in MPS IVA patients were significantly higher than those in age-matched controls (0.67-4.6 µg/ml; P < 0.0001). It was found that blood KS level varied with age and clinical severity in the patients. Blood KS levels in MPS IVA peaked between 2 years and 5 years of age (mean 11.4 µg/ml). Blood KS levels in severe MPS IVA (mean 7.3 µg/ml) were higher than in the attenuated form (mean 2.1 µg/ml) (P = 0.012). We also found elevated blood KS levels in other types of MPS. These findings indicate that the new KS assay for blood is suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Late-Onset Ornithine Transcarbamylase Deficiency and Variable Phenotypes in Vietnamese Females With OTC Mutations.

Background: Ornithine transcarbamylase deficiency (OTCD) is an X- linked recessive disorder and the most common error of the urea cycle, caused by the mutations in the OTC gene. Due to X-inactivation, 15-20% of female carriers present symptoms of OTCD at late onset. Early diagnosis of OTCD by molecular analysis in females is highly desirable. The aim of the study was to identify the mutations in two unrelated Vietnamese girls suspected with OTCD and the carriers in their families for definitive diagnosis and proper counseling. Case Presentation: Two patients presented with an acute encephalopathy at the first admission. Biochemical tests revealed hyperammonemia, hyperlactatemia, elevated glutamine level, elevated transaminase, elevated urinary orotic and uracil acid levels, and disorder of prothrombin time. Brain magnetic resonance imaging indicated cerebral edema. Based on the clinical and laboratory results, the two patients were diagnosed with urea cycle disorders. Therefore, the two patients were managed by stopping feeding, with infused glucose, l-carnitine, l-arginine, and sodium benzoate, and with hemofiltration. The two patients were alert and recovered with normal blood ammonia levels after 72 h of treatment. The family history of patient 1 showed that her brother died at 4 days of age due to a coma and dyspnea, while her parents were asymptomatic. Variable phenotypes were observed in three generations of the patient 2's family, including asymptomatic (mother), affected female adults dying at the first symptom (grandmother and aunt), and affected males dying in the first week of life (uncle, cousin, and siblings). Whole-exome sequencing showed two mutations in the OTC gene, including one novel missense mutation, c.365A>T, in the patient 1 and one previously reported splicing mutation, c.717+1G>A, in the patient 2. The two mutations are evaluated as likely pathogenic and pathogenic, respectively, according to the recommendations of the American College of Medical Genetics and Genomics (ACMG). Genetic analyses in the families indicated the mothers were heterozygous. Conclusion: Clinical, biochemical, and molecular findings accurately diagnosed the two patients with late-onset OTCD. Our results explained the genetic causes and proposed the risk in the patients' families, which could be useful for genetic counseling and monitoring in prenatal diagnosis.

A novel frameshift PHKA2 mutation in a family with glycogen storage disease type IXa: A first report in Vietnam and review of literature.

BACKGROUND: Glycogen storage diseases (GSDs) are clinically and genetically heterogeneous disorders. Overlapping features between liver GSDs are a major challenge in the clinical diagnosis of them. Genetic testing can provide an early and accurate diagnosis of patients suspected with GSDs. CASE PRESENTATION: In this study, we report two siblings born to healthy, non-consanguineous Vietnamese parents with hepatomegaly. The proband presented with hepatomegaly, normal spleen, elevated transaminases, without hypoglycemia, normal lactate dehydrogenase and creatine kinase. Liver biopsy revealed degeneration and swollen hepatocytes, suggesting a diagnosis with GSDs. METHODS: Whole exome sequencing was applied to identify genetic variants in the proband. Variant validation and familial co-segregation analysis were examined using Sanger sequencing. RESULTS: A novel frameshift duplication mutation c.3308_3312dupATGTC (p.L1105Mfs*11) of the PHKA2 gene was identified in the proband and his elder brother at the hemizygous state. This mutation was inherited from their mother. Their father and younger brother were normal genotype. CONCLUSIONS: The two siblings were accurately diagnosed with GSD type XIa. This is the first case report of GSD type IXa in Vietnamese patients with a mutation in the PHKA2 gene. This finding may support for genetics diagnosis of unknown cause of hepatomegaly.

De Novo Mutations in EIF2B1 Affecting eIF2 Signaling Cause Neonatal/Early-Onset Diabetes and Transient Hepatic Dysfunction.

Permanent neonatal diabetes mellitus (PNDM) is caused by reduced ß-cell number or impaired ß-cell function. Understanding of the genetic basis of this disorder highlights fundamental ß-cell mechanisms. We performed trio genome sequencing for 44 patients with PNDM and their unaffected parents to identify causative de novo variants. Replication studies were performed in 188 patients diagnosed with diabetes before 2 years of age without a genetic diagnosis. EIF2B1 (encoding the eIF2B complex α subunit) was the only gene with novel de novo variants (all missense) in at least three patients. Replication studies identified two further patients with de novo EIF2B1 variants. In addition to having diabetes, four of five patients had hepatitis-like episodes in childhood. The EIF2B1 de novo mutations were found to map to the same protein surface. We propose that these variants render the eIF2B complex insensitive to eIF2 phosphorylation, which occurs under stress conditions and triggers expression of stress response genes. Failure of eIF2B to sense eIF2 phosphorylation likely leads to unregulated unfolded protein response and cell death. Our results establish de novo EIF2B1 mutations as a novel cause of permanent diabetes and liver dysfunction. These findings confirm the importance of cell stress regulation for ß-cells and highlight EIF2B1's fundamental role within this pathway.