De novo NIPBL Mutations in Vietnamese Patients with Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a rare congenital genetic disease causing abnormal unique facial phenotypes, several defects in organs and body parts, and mental disorder or intellectual disorder traits. Main causes of CdLS have been reported as variants in cohesin complex genes, in which mutations in the NIPBL gene have been estimated to account for up to 80%. Our study included three Vietnamese patients with typical CdLS phenotypes. Whole exome sequencing revealed two known heterozygous mutations c.6697G>A (p.Val2233Met) and c.2602C>T (p.Arg868X), and a novel heterozygous mutation c.4504delG (p.Val1502fsX87) in the NIPBL gene of the three patients. In silico analyses of the identified mutations predicted possible damaging and truncating effects on the NIPBL protein. Inherited analyses in the patients' families showed that all of the mutations are de novo. Our results lead a definitive diagnosis of patients with CdLS and expand the spectrum of mutations in the NIPBL gene. These findings also confirm whole exome sequencing is an efficient tool for genetic screening of CdLS.

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.

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.

Identification of three novel mutations in PCNT in vietnamese patients with microcephalic osteodysplastic primordial dwarfism type II.

BACKGROUND: Primordial dwarfism (PD) is a group of genetically heterogeneous disorders related to developmental disabilities occurring in the uterus and prolongs during all stages of life, resulting in short stature, facial deformities and abnormal brain. OBJECTIVE: To determine the exact cause of the disease in two Vietnamese patients priory diagnosed with PD by severe pre-and postnatal growth retardation with marked microcephaly and some bone abnormalities. METHODS: Whole-exome sequencing was performed for the two patients and mutations in genes related to PD were screened. Sanger sequencing was applied to examine the mutations in the patients of their families. RESULTS: Three novel mutations in the PCNT gene which have not been reported previously were identified in the two patients. Of which, two frameshift mutations (p.Thr479Profs*6 and p.Glu2742Alafs*8) were detected in patient I and one stop-gained mutation (p.Gln1907*) was detected in the patient II. These mutations may result in a truncated PCNT protein, leading to an inactivated PACT domain corresponding to residue His3138-Trp3216 of PCNT protein. Therefore, the three mutations may cause a deficiency of protein functional activity and result in the phenotypes of primordial dwarfism in the two patients. CONCLUSIONS: Clinical presentations in combination with genetic analyses supported an accurate diagnosis of the two patients with microcephalic osteodysplastic primordial dwarfism type II (MOPD II). In addition, these results have important implications for prenatal genetic screening and genetic counseling for the families.

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.

Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Whole Exome Sequencing as a Diagnostic Tool for Unidentified Muscular Dystrophy in a Vietnamese Family.

Muscular dystrophies are a group of heterogeneous clinical and genetic disorders. Two siblings presented with characteristics like muscular dystrophy, abnormal white matter, and elevated serum creatine kinase level. The high throughput of whole exome sequencing (WES) makes it an efficient tool for obtaining a precise diagnosis without the need for immunohistochemistry. WES was performed in the two siblings and their parents, followed by prioritization of variants and validation by Sanger sequencing. Very rare variants with moderate to high predicted impact in genes associated with neuromuscular disorders were selected. We identified two pathogenic missense variants, c.778C>T (p.H260Y) and c.2987G>A (p.C996Y), in the LAMA2 gene (NM_000426.3), in the homozygous state in two siblings, and in the heterozygous state in their unaffected parents, which were confirmed by Sanger sequencing. Variant c.2987G>A has not been reported previously. These variants may lead to a change in the structure and function of laminin-α2, a member of the family of laminin-211, which is an extracellular matrix protein that functions to stabilize the basement membrane of muscle fibers during contractions. Overall, WES enabled an accurate diagnosis of both patients with LAMA2-related muscular dystrophy and expanded the spectrum of missense variants in LAMA2.

An ABCC8 Nonsense Mutation Causing Neonatal Diabetes Through Altered Transcript Expression.

The pancreatic ATP-sensitive K+ (K-ATP) channel is a key regulator of insulin secretion. Gain-of-function mutations in the genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the channel cause neonatal diabetes, whilst loss-of-function mutations in these genes result in congenital hyperinsulinism. We report two patients with neonatal diabetes in whom we unexpectedly identified recessively inherited loss-of-function mutations. The aim of this study was to investigate how a homozygous nonsense mutation in ABCC8 could result in neonatal diabetes. The ABCC8 p.Glu747* was identified in two unrelated Vietnamese patients. This mutation is located within the in-frame exon 17 and RNA studies confirmed (a) the absence of full length SUR1 mRNA and (b) the presence of the alternatively spliced transcript lacking exon 17. Successful transfer of both patients to sulphonylurea treatment suggests that the altered transcript expression enhances the sensitivity of the K-ATP channel to Mg-ADP/ATP. This is the first report of an ABCC8 nonsense mutation causing a gain-of-channel function and these findings extend the spectrum of K-ATP channel mutations observed in patients with neonatal diabetes.