Julia Vodopiutz

Loss of Dermatan-4-Sulfotransferase 1 Function Results in Adducted Thumb-Clubfoot Syndrome

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-alpha1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patients fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance.

An overview of L‐2‐hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype–phenotype study

L‐2‐Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2‐hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT‐PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty‐five novel mutations as well as 35 reported mutations and 14 nondisease‐related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype–genotype relationship. Hum Mutat 30:1–11, 2010.

Loss of dermatan sulfate epimerase (DSE) function results in musculocontractural Ehlers–Danlos syndrome

The sulfated polysaccharide dermatan sulfate (DS) forms proteoglycans with a number of distinct core proteins. Iduronic acid-containing domains in DS have a key role in mediating the functions of DS proteoglycans. Two tissue-specific DS epimerases, encoded by DSE and DSEL, and a GalNAc-4-O-sulfotransferase encoded by CHST14 are necessary for the formation of these domains. CHST14 mutations were previously identified for patients with the musculocontractural type of Ehlers-Danlos syndrome (MCEDS). We now identified a homozygous DSE missense mutation (c.803C>T, p.S268L) by the positional candidate approach in a male child with MCEDS, who was born to consanguineous parents. Heterologous expression of mutant full-length and soluble recombinant DSE proteins showed a loss of activity towards partially desulfated DS. Patient-derived fibroblasts also showed a significant reduction in epimerase activity. The amount of DS disaccharides was markedly decreased in the conditioned medium and the cell fraction from cultured fibroblasts of the patient when compared with a healthy control subject, whereas no apparent difference was observed in the chondroitin sulfate (CS) chains from the conditioned media. However, the total amount of CS disaccharides in the cell fraction from the patient was increased ∼1.5-fold, indicating an increased synthesis or a reduced conversion of CS chains in the cell fraction. Stable transfection of patient fibroblasts with a DSE expression vector increased the amount of secreted DS disaccharides. DSE deficiency represents a specific defect of DS biosynthesis. We demonstrate locus heterogeneity in MCEDS and provide evidence for the importance of DS in human development and extracellular matrix maintenance.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C

Objective Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. Design We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. Results We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. Conclusions Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD.

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

Defining the Phenotype in Congenital Disorder of Glycosylation Due to ALG1 Mutations

Deficiency of β-1,4 mannosyltransferase (MT-1) congenital disorder of glycosylation (CDG), due to ALG1 gene mutations. Features in 9 patients reported previously consisted of prenatal growth retardation, pregnancy-induced maternal hypertension and fetal hydrops. Four patients died before 5 years of age, and survivors showed a severe psychomotor retardation. We report on 7 patients with psychomotor delay, microcephaly, strabismus and coagulation abnormalities, seizures and abnormal fat distribution. Four children had a stable clinical course, two had visual impairment, and 1 had hearing loss. Thrombotic and vascular events led to deterioration of the clinical outcome in 2 patients. Four novel ALG1 mutations were identified. Pathogenicity was determined in alg1 yeast mutants transformed with hALG1. Functional analyses showed all novel mutations representing hypomorphs associated with residual enzyme activity. We extend the phenotypic spectrum including the first description of deafness in MT1 deficiency, and report on mildly affected patients, surviving to adulthood. The dysmorphic features, including abnormal fat distribution and strabismus highly resemble CDG due to phosphomannomutase-2 deficiency (PMM2-CDG), the most common type of CDG. We suggest testing for ALG1 mutations in unsolved CDG patients with a type 1 transferrin isoelectric focusing pattern, especially with epilepsy, severe visual loss and hemorrhagic/thrombotic events.

Hidden Mutations in Cornelia de Lange Syndrome Limitations of Sanger Sequencing in Molecular Diagnostics

Cornelia de Lange syndrome (CdLS) is a well‐characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high‐coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.

WDR73 Mutations Cause Infantile Neurodegeneration and Variable Glomerular Kidney Disease

Infantile‐onset cerebellar atrophy (CA) is a clinically and genetically heterogeneous trait. Galloway–Mowat syndrome (GMS) is a rare autosomal recessive disease, characterized by microcephaly with brain anomalies including CA in some cases, intellectual disability, and early‐infantile‐onset nephrotic syndrome. Very recently, WDR73 deficiency was identified as the cause of GMS in five individuals. To evaluate the role of WDR73 mutations as a cause of GMS and other forms of syndromic CA, we performed Sanger or exome sequencing in 51 unrelated patients with CA and variable brain anomalies and in 40 unrelated patients with a diagnosis of GMS. We identified 10 patients from three CA and from two GMS families with WDR73 mutations including the original family described with CA, mental retardation, optic atrophy, and skin abnormalities (CAMOS). There were five novel mutations, of which two were truncating and three were missense mutations affecting highly conserved residues. Individuals carrying homozygous WDR73 mutations mainly presented with a pattern of neurological and neuroimaging findings as well as intellectual disability, while kidney involvement was variable. We document postnatal onset of CA, a retinopathy, basal ganglia degeneration, and short stature as novel features of WDR73‐related disease, and define WDR73‐related disease as a new entity of infantile neurodegeneration.

Congenital disorders of glycosylation—a challenging group of IEMs

SummaryCongenital disorders of glycosylation (CDG) are a rapidly growing group of inherited errors of metabolism (IEMs) due to an impairment of one or several glycosylation pathways. During recent years over 30 CDG subtypes have been identified at a molecular and biochemical level. The clinical manifestations in CDG are heterogeneous and may be highly variable within the same subtype and even among affected siblings. Novel insights into the extremely complex glycosylation pathways have necessitated several reclassifications of the group of CDG. Today CDG comprise not only the formerly known multisystem glycosylation defects but also some tissue-specific glycosylation defects, implicating a different diagnostic work-up depending on the underlying glycosylation defect. In 2007 the expanding group of CDG is an enormous challenge to all specialists working in the field of IEMs. This review gives a brief overview about the expanded group of CDG and summarizes the main implications for clinicians.