ALG11‐CDG syndrome: Expanding the phenotype

ALG11‐Congenital Disorder of Glycosylation (ALG11‐CDG, also known as congenital disorder of glycosylation type Ip) is an inherited inborn error of metabolism due to abnormal protein and lipid glycosylation. We describe two unrelated patients with ALG11‐CDG due to novel mutations, review the literature of previously described affected individuals, and further expand the clinical phenotype. Both affected individuals reported here had severe psychomotor disabilities and epilepsy. Their fibroblasts synthesized truncated precursor glycan structures, consistent with ALG11‐CDG, while also showing hypoglycosylation of a novel biomarker, GP130. Surprisingly, one patient presented with normal transferrin glycosylation profile, a feature that has not been reported previously in patients with ALG11‐CDG. Together, our data expand the clinical and mutational spectrum of ALG11‐CDG.     

Exome sequence identified a c.320A > G ALG13 variant in a female with infantile epileptic encephalopathy with normal glycosylation and random X inactivation: Review of the literature

Congenital Disorders of Glycosylation (CDG) are new and rapidly expanding neurometabolic disorders with multisystem involvements, broad phenotypic manifestations, and variable severity. The majority results from a defect of one of the steps involved with protein or lipid N-glycosylation pathway. Almost all are inherited in autosomal recessive patterns with a few exceptions such as the X-linked ALG13. Mutations of ALG13 are reported, so far in only 10 patients, all were ascertained through exome/genome sequencing. Specifically, the ALG13 c.320A > G (p.Asn107Ser) variant was reported only in females and in all were de novo mutations. These findings may suggest an X-linked dominant inheritance of this mutation with embryonic male lethality. These patients presented with severe infantile epileptic encephalopathy, global developmental delay, and multisystem abnormalities. Only two of these females had glycosylation studies done, and both showed normal pattern of glycosylated serum transferrin isoforms, and none had their X-chromosome inactivation patterns studied.Here, we report on another female patient who is heterozygous for the same ALG13 c.320A > G (p.Asn107Ser) variant. She presented with infantile spasms, epileptic encephalopathy, hypsarrhythmia, hypotonia, developmental delay, intellectual disability, abnormal coagulation profile, feeding problems, hypotonia, and dysmorphic features. The diagnosis of CGD was suspected clinically, but glycosylation studies were done twice and showed normal patterns on both occasions. Her X-inactivation study was also done and, surprisingly, showed a random pattern of X-inactivation, with no evidence of skewness.     

CSGALNACT1‐congenital disorder of glycosylation: A mild skeletal dysplasia with advanced bone age

Congenital disorders of glycosylation (CDGs) comprise a large number of inherited metabolic defects that affect the biosynthesis and attachment of glycans. CDGs manifest as a broad spectrum of disease, most often including neurodevelopmental and skeletal abnormalities and skin laxity. Two patients with biallelic CSGALNACT1 variants and a mild skeletal dysplasia have been described previously. We investigated two unrelated patients presenting with short stature with advanced bone age, facial dysmorphism, and mild language delay, in whom trio‐exome sequencing identified novel biallelic CSGALNACT1 variants: compound heterozygosity for c.1294G>T (p.Asp432Tyr) and the deletion of exon 4 that includes the start codon in one patient, and homozygosity for c.791A>G (p.Asn264Ser) in the other patient. CSGALNACT1 encodes CSGalNAcT‐1, a key enzyme in the biosynthesis of sulfated glycosaminoglycans chondroitin and dermatan sulfate. Biochemical studies demonstrated significantly reduced CSGalNAcT‐1 activity of the novel missense variants, as reported previously for the p.Pro384Arg variant. Altered levels of chondroitin, dermatan, and heparan sulfate moieties were observed in patients’ fibroblasts compared to controls. Our data indicate that biallelic loss‐of‐function mutations in CSGALNACT1 disturb glycosaminoglycan synthesis and cause a mild skeletal dysplasia with advanced bone age, CSGALNACT1‐CDG.     

Could distal variants in ALG13 lead to atypical clinical presentation?

Congenital disorders of glycosylation (CDG) represent a wide range of some 150 inherited metabolic diseases, continually expanding in terms of newly identified genes and the heterogeneity of clinical and molecular presentations within each subtype. Heterozygous pathogenic variants in ALG13 are associated with early-onset epileptic encephalopathy, typically in females. The majority of subjects described so far harbour one of the two recurrent pathogenic variants, namely p.(Asn107Ser) and p.(Ala81Thr) in the C-terminal glycosyltransferase domain. We report a novel ALG13 variant (c.1709G > A, p.(Gly570Glu)) in an adult female with unremarkable past developmental and medical history, except for mild kinetic tremor. Our proband presented with acute onset of neurological and psychiatric features, along with liver dysfunction, during pregnancy, all of which gradually resolved after delivery. The proband's newborn baby died at 22 days of life from neonatal liver disease, due to gestational alloimmune liver disease (GALD). Functional assessment on fibroblasts derived from our case showed alterations in 2 of 3 cellular glycosylation markers (LAMP2, Factor IX), suggesting a functional effect of this novel ALG13 variant on glycosylation. This paper raises the possibility that variants outside the glycosyltransferase domain may have a hypomorphic effect leading to atypical clinical manifestations.     

RFT1 deficiency in three novel CDG patients

The medical significance of N‐glycosylation is underlined by a group of inherited human disorders called Congenital Disorders of Glycosylation (CDG). One key step in the biosynthesis of the Glc₃Man₉GlcNAc₂‐PP‐dolichol precursor, essential for N‐glycosylation, is the translocation of Man₅GlcNAc₂‐PP‐dolichol across the endoplasmic reticulum membrane. This step is facilitated by the RFT1 protein. Recently, the first RFT1‐deficient CDG (RFT1‐CDG) patient was identified and presented a severe N‐glycosylation disorder. In the present study, we describe three novel CDG patients with an RFT1 deficiency. The first patient was homozygous for the earlier reported RFT1 missense mutation (c.199C>T; p.R67C), whereas the two other patients were homozygous for the missense mutation c.454A>G (p.K152E) and c.892G>A (p.E298 K), respectively. The pathogenic character of the novel mutations was illustrated by the accumulation of Man₅GlcNAc₂‐PP‐dolichol and by reduced recombinant DNase 1 secretion. Both the glycosylation pattern and recombinant DNase 1 secretion could be normalized by expression of normal RFT1 cDNA in the patients' fibroblasts. The clinical phenotype of these patients comprised typical CDG symptoms in addition to sensorineural deafness, rarely reported in CDG patients. The identification of additional RFT1‐deficient patients allowed to delineate the main clinical picture of RFT1‐CDG and confirmed the crucial role of RFT1 in Man₅GlcNAc₂‐PP‐dolichol translocation. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is one of the most common X‐linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity‐confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.     

Ten novel HMGCL mutations in 24 patients of different origin with 3‐hydroxy‐3‐methyl‐glutaric aciduria

3‐Hydroxy‐3‐methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L‐leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes.” © 2009 Wiley‐Liss, Inc.     

Severe ALG8-CDG (CDG-Ih) associated with homozygosity for two novel missense mutations detected by exome sequencing of candidate genes

Posttranslationally glycosylated proteins are important in many biological processes in humans and Congenital disorders of glycosylation (CDGs) are associated with a broad range of phenotypes. Type I CDGs are a group of rare autosomal recessive conditions. To date 17 subtypes have been enzymatically and molecularly characterized. Impaired function of the enzyme dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase encoded by the ALG8 gene, causes ALG8-CDG (CDG-Ih, OMIM #608104). This enzyme facilitates the transfer of a second glucose molecule to a growing lipid-linked oligosaccharide chain, a process that transpires in the endoplasmic reticulum (ER). We present a female patient of consanguineous parents, with pre- and postnatal growth retardation, dysmorphic features, significant developmental delay, visual impairment and an electrophoretic serum transferrin pattern indicative of a type I CDG. Type I CDG subgroup was determined by exome sequencing facilitated by homozygosity analysis. The patient was homozygous for two variants, nine nucleotides apart, in exon 8 of ALG8; c.799T > C [p.Ser267Pro] and c.808T > C [p.Phe270Leu]. Both missense mutations are predicted to affect a conserved region of an intraluminal ER loop of dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase. To our knowledge, the current report describes the ninth published case of ALG8-CDG, contributing to the further delineation of this rare and variable disorder.     

Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc‐1‐Phosphotransferase

The lysosomal storage disorder ML III γ is caused by defects in the γ subunit of UDP‐GlcNAc:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase, the enzyme that tags lysosomal enzymes with the mannose 6‐phosphate lysosomal targeting signal. In patients with this disorder, most of the newly synthesized lysosomal enzymes are secreted rather than being sorted to lysosomes, resulting in increased levels of these enzymes in the plasma. Several missense mutations in GNPTG, the gene encoding the γ subunit, have been reported in mucolipidosis III γ patients. However, in most cases, the impact of these mutations on γ subunit function has remained unclear. Here, we report that the variants c.316G>A (p.G106S), c.376G>A (p.G126S), and c.425G>A (p.C142Y) cause misfolding of the γ subunit, whereas another variant, c.857C>T (p.T286M), does not appear to alter γ subunit function. The misfolded γ subunits were retained in the ER and failed to rescue the lysosomal targeting of lysosomal acid glycosidases.     

Functional characterization of four ATP‐binding cassette transporter A3 gene (ABCA3) variants

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase‐mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild‐type (WT‐ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body‐like vesicle diameter and intracellular ABCA3 trafficking using fluorescence‐based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well‐organized lamellar body‐like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well‐organized lamellar body‐like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT‐ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.     

Functional analysis of three splicing mutations identified in the PMM2 gene: Toward a new therapy for congenital disorder of glycosylation type Ia

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N‐glycosylation. The most prevalent form of CDG—type Ia—is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256–1G>C and c.640–9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640–15479C>T). Cell‐based splicing assays strongly suggest that all these are disease‐causing splicing mutations. The c.256–1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640–9T>G mutation was found responsible for the activation of a cryptic intronic splice‐site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine‐rich (SR) proteins. Finally, the deep intronic change c.640–15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation‐specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic

Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6. This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway. CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties. We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6. Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p. Ala333Val), which occurs in over half of all known CDG-Ic patients. The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200. We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6. However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal. Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms. Genotyping additional patients with CDG-like symptoms will be required to resolve this issue.     

Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan‐α‐glucosaminide N‐acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease‐causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice‐site mutation c.494?1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372?2G>A, and c.234+1G>A present in cis with c.564‐98T>C and c.710C>A rare single‐nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease‐causing variants, and reveal the evolutionary history of MPSIIIC.     

Identifying haplotypes in recessive inherited retinal dystrophies using whole‐genome linked‐read sequencing

Conventional next‐generation sequencing methods, used in most gene panels, cannot separate maternally and paternally derived sequence information of distant variants. In recessive diseases, two or more equally plausible causative variants with unsolved phase information prevent accurate molecular diagnosis. In reality, close relatives might be unavailable for segregation analysis. Here, we utilized whole genome linked‐read sequencing to assign variants to haplotypes in two patients with inherited retinal dystrophies. Patient 1 with macular dystrophy had variants c.3442T>C, p.(Cys1148Arg), c.4209G>T, p.(Glu1403Asp), and c.1182C>T, p.(Cys394=) in CRB1, and Patient 2 with nonsyndromic retinitis pigmentosa had c.1328T>A, p.(Val443Asp) and c.3032C>G, p.(Ser1011*) in AHI1. The relatives were not available for genotyping. Using whole genome linked‐read sequencing we phased the variants to haplotypes providing genetic background for the retinal dystrophies. In future, when the price of sequencing methods that provides long‐read data decreases and their read‐depth and accuracy increases, they are probably considered the primary or adjunctive sequencing methods in genetic testing, allowing the immediate collection of phase information and thus obviating the need for the carrier testing and segregation analysis.