The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter

Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.     

Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2

We identified loss-of-function mutations in ATP6V0A2, encoding the a2 subunit of the V-type H+ ATPase, in several families with autosomal recessive cutis laxa type II or wrinkly skin syndrome. The mutations result in abnormal glycosylation of serum proteins (CDG-II) and cause an impairment of Golgi trafficking in fibroblasts from affected individuals. These results indicate that the a2 subunit of the proton pump has an important role in Golgi function.     

Mutations in MVK, encoding mevalonate kinase, cause hyperimmunoglobulinaemia D and periodic fever syndrome.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash. Diagnostic hallmark of HIDS is a constitutively elevated level of serum immunoglobulin D (IgD), although patients have been reported with normal IgD levels. To determine the underlying defect in HIDS, we analysed urine of several patients and discovered increased concentrations of mevalonic acid during severe episodes of fever, but not between crises. Subsequent analysis of cells from four unrelated HIDS patients revealed reduced activities of mevalonate kinase (MK; encoded by the gene MVK), a key enzyme of isoprenoid biosynthesis. Sequence analysis of MVK cDNA from the patients identified three different mutations, one of which was common to all patients. Expression of the mutant cDNAs in Escherichia coli showed that all three mutations affect the activity of the encoded proteins. Moreover, immunoblot analysis demonstrated a deficiency of MK protein in patient fibroblasts, indicating a protein-destabilizing effect of the mutations.     

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.     

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.     

Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.     

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein alphaA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.     

The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy

Disorganization of the neurofilament network is a prominent feature of several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy and axonal Charcot-Marie-Tooth disease. Giant axonal neuropathy (GAN, MIM 256850), a severe, autosomal recessive sensorimotor neuropathy affecting both the peripheral nerves and the central nervous system, is characterized by neurofilament accumulation, leading to segmental distension of the axons. GAN corresponds to a generalized disorganization of the cytoskeletal intermediate filaments (IFs), to which neurofilaments belong, as abnormal aggregation of multiple tissue-specific IFs has been reported: vimentin in endothelial cells, Schwann cells and cultured skin fibroblasts, and glial fibrillary acidic protein (GFAP) in astrocytes. Keratin IFs also seem to be alterated, as most patients present characteristic curly or kinky hairs. We report here identification of the gene GAN, which encodes a novel, ubiquitously expressed protein we have named gigaxonin. We found one frameshift, four nonsense and nine missense mutations in GAN of GAN patients. Gigaxonin is composed of an amino-terminal BTB (for Broad-Complex, Tramtrack and Bric a brac) domain followed by a six kelch repeats, which are predicted to adopt a beta-propeller shape. Distantly related proteins sharing a similar domain organization have various functions associated with the cytoskeleton, predicting that gigaxonin is a novel and distinct cytoskeletal protein that may represent a general pathological target for other neurodegenerative disorders with alterations in the neurofilament network.     

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.     

Congenital adrenal hyperplasia due to point mutations in the type II 3 beta-hydroxysteroid dehydrogenase gene.

Classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) deficiency is an autosomal recessive form of congenital adrenal hyperplasia characterized by a severe impairment of steroid biosynthesis in both the adrenals and the gonads. We describe the nucleotide sequence of the two highly homologous genes encoding 3 beta-HSD isoenzymes in three classic 3 beta-HSD deficient patients belonging to two apparently unrelated pedigrees. No mutation was detected in the type I 3 beta-HSD gene, which is mainly expressed in the placenta and peripheral tissues. Both nonsense and frameshift mutations, however, were found in the type II 3 beta-HSD gene, which is the predominant 3 beta-HSD gene expressed in the adrenals and gonads, thus providing the first elucidation of the molecular basis of this disorder.     

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.     

ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis

The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis, but it is also suspected to contribute to anticancer drug resistance, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(-) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(-) (ABCB6(-/-)) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.     

A homozygous stop codon in the lysyl hydroxylase gene in two siblings with Ehlers-Danlos syndrome type VI.

Ehlers-Danlos syndrome (EDS) is characterized by joint hypermobility, alterations in the skin and additional signs of connective tissue involvement. EDS type VI was the first connective tissue disorder for which a specific defect in collagen metabolism was identified, namely a deficiency of lysyl hydroxylase activity. We now report a homozygous single basepair substitution converting the CGA codon (Arg319) to a TGA termination codon in two siblings with EDS type VI. The healthy parents, who are first cousins, and two of the three healthy siblings of the patients are heterozygous. The mutation leads to an almost complete absence of lysyl hydroxylase activity in extracts derived from fibroblasts of the patients.     

Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.     

Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA)

Iron deficiency is usually attributed to chronic blood loss or inadequate dietary intake. Here, we show that iron deficiency anemia refractory to oral iron therapy can be caused by germline mutations in TMPRSS6, which encodes a type II transmembrane serine protease produced by the liver that regulates the expression of the systemic iron regulatory hormone hepcidin. These findings demonstrate that TMPRSS6 is essential for normal systemic iron homeostasis in humans.     

Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome.

Thiamine-responsive megaloblastic anaemia syndrome (TRMA; MIM 249270) is an autosomal recessive disorder with features that include megaloblastic anaemia, mild thrombocytopenia and leucopenia, sensorineural deafness and diabetes mellitus. Treatment with pharmacologic doses of thiamine ameliorates the megaloblastic anaemia and diabetes mellitus. A defect in the plasma membrane transport of thiamine has been demonstrated in erythrocytes and cultured skin fibroblasts from TRMA patients. The gene causing TRMA was assigned to 1q23.2-q23.3 by linkage analysis. Here we report the cloning of a new gene, SLC19A2, identified from high-through-put genomic sequences due to homology with SLC19A1, encoding reduced folate carrier 1 (refs 8-10). We cloned the entire coding region by screening a human fetal brain cDNA library. SLC19A2 encodes a protein (of 497 aa) predicted to have 12 transmembrane domains. We identified 2 frameshift mutations in exon 2. a 1-bp insertion and a 2-bp deletion, among four Iranian families with TRMA. The sequence homology and predicted structure of SLC19A2, as well as its role in TRMA, suggest that its gene product is a thiamine carrier, the first to be identified in complex eukaryotes.     

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.     

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.     

Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But approximately 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.