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.     

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.     

A putative RUNX1 binding site variant between SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis

Psoriasis (OMIM 177900) is a chronic inflammatory skin disorder of unknown pathogenesis affecting approximately 2% of the Western population. It occurs more frequently in individuals with human immunodeficiency virus, and 20-30% of individuals with psoriasis have psoriatic arthritis. Psoriasis is associated with HLA class I alleles, and previous linkage analysis by our group identified a second psoriasis locus at 17q24-q25 (PSORS2; ref. 7). Linkage to this locus was confirmed with independent family sets. Additional loci have also been proposed to be associated with psoriasis. Here we describe two peaks of strong association with psoriasis on chromosome 17q25 separated by 6 Mb. Associated single-nucleotide polymorphisms (SNPs) in the proximal peak lie in or near SLC9A3R1 (also called EBP50 and NHERF1) and NAT9, a new member of the N-acetyltransferase family. SLC9A3R1 is a PDZ domain-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family and is implicated in diverse aspects of epithelial membrane biology and immune synapse formation in T cells. The distal peak of association is in RAPTOR (p150 target of rapamycin (TOR)-scaffold protein containing WD-repeats). Expression of SLC9A3R1 is highest in the uppermost stratum Malpighi of psoriatic and normal skin and in inactive versus active T cells. A disease-associated SNP lying between SLC9A3R1 and NAT9 leads to loss of RUNX1 binding. This is the second example of loss of a RUNX1 binding site associated with susceptibility to an autoimmune disease. It also suggests defective regulation of SLC9A3R1 or NAT9 by RUNX1 as a susceptibility factor for psoriasis.     

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.     

A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases

Sialic acid storage diseases (SASD, MIM 269920) are autosomal recessive neurodegenerative disorders that may present as a severe infantile form (ISSD) or a slowly progressive adult form, which is prevalent in Finland (Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation; visceromegaly and coarse features are also present in infantile cases. Progressive cerebellar atrophy and dysmyelination have been documented by magnetic resonance imaging (ref. 4). Enlarged lysosomes are seen on electron microscopic studies and patients excrete large amounts of free sialic acid in urine. A H+/anionic sugar symporter mechanism for sialic acid and glucuronic acid is impaired in lysosomal membranes from Salla and ISSD patients. The locus for Salla disease was assigned to a region of approximately 200 kb on chromosome 6q14-q15 in a linkage study using Finnish families. Salla disease and ISSD were further shown to be allelic disorders. A physical map with P1 and PAC clones was constructed to cover the 200-kb area flanked by the loci D6S280 and D6S1622, providing the basis for precise physical positioning of the gene. Here we describe a new gene, SLC17A5 (also known as AST), encoding a protein (sialin) with a predicted transport function that belongs to a family of anion/cation symporters (ACS). We found a homozygous SLC17A5 mutation (R39C) in five Finnish patients with Salla disease and six different SLC17A5 mutations in six ISSD patients of different ethnic origins. Our observations suggest that mutations in SLC17A5 are the primary cause of lysosomal sialic acid storage diseases.     

Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin

Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.     

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.     

Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis

Variants associated with meconium ileus in cystic fibrosis were identified in 3,763 affected individuals by genome-wide association study (GWAS). Five SNPs at two loci near SLC6A14 at Xq23-24 (minimum P = 1.28 × 10(-12) at rs3788766) and SLC26A9 at 1q32.1 (minimum P = 9.88 × 10(-9) at rs4077468) accounted for ~5% of phenotypic variability and were replicated in an independent sample of affected individuals (n = 2,372; P = 0.001 and 0.0001, respectively). By incorporating the knowledge that disease-causing mutations in CFTR alter electrolyte and fluid flux across surface epithelium into a hypothesis-driven GWAS (GWAS-HD), we identified associations with the same SNPs in SLC6A14 and SLC26A9 and established evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple genes encoding constituents of the apical plasma membrane where CFTR resides (P = 0.0002; testing of 155 apical membrane genes jointly and in replication, P = 0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis.     

Heterozygous TGFBR2 mutations in Marfan syndrome

Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin (FBN1) at 15q21.1 (ref. 1). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2, at 3p25-p24.2 in a large French family (family MS1). Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-beta receptor 2 (TGFBR2) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G-->A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-beta signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.     

Mutations in KERA, encoding keratocan, cause cornea plana

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.     

Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2

The autosomal recessive form of Robinow syndrome (RRS; MIM 268310) is a severe skeletal dysplasia with generalized limb bone shortening, segmental defects of the spine, brachydactyly and a dysmorphic facial appearance. We previously mapped the gene mutated in RRS to chromosome 9q22 (ref. 4), a region that overlaps the locus for autosomal dominant brachydactyly type B (refs 5,6). The recent identification of ROR2, encoding an orphan receptor tyrosine kinase, as the gene mutated in brachydactyly type B (BDB1; ref. 7) and the mesomelic dwarfing in mice homozygous for a lacZ and/or a neo insertion into Ror2 (refs 8,9) made this gene a candidate for RRS. Here we report homozygous missense mutations in both intracellular and extracellular domains of ROR2 in affected individuals from 3 unrelated consanguineous families, and a nonsense mutation that removes the tyrosine kinase domain and all subsequent 3' regions of the gene in 14 patients from 7 families from Oman. The nature of these mutations suggests that RRS is caused by loss of ROR2 activity. The identification of mutations in three distinct domains (containing Frizzled-like, kringle and tyrosine kinase motifs) indicates that these are all essential for ROR2 function.     

Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4-13). These dominant forms of MED (EDM1-3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1-3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24-p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.     

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.     

Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b-/- mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.     

Heterozygous missense mutations in BSCL2 are associated with distal hereditary motor neuropathy and Silver syndrome

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12-q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.     

Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I

Hereditary sensory neuropathy type I (HSN1) is the most common hereditary disorder of peripheral sensory neurons. HSN1 is an autosomal dominant progressive degeneration of dorsal root ganglia and motor neurons with onset in the second or third decades. Initial symptoms are sensory loss in the feet followed by distal muscle wasting and weakness. Loss of pain sensation leads to chronic skin ulcers and distal amputations. The HSN1 locus has been mapped to chromosome 9q22.1-22.3 (refs. 3,4). Here we map the gene SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, to this locus. Mutation screening revealed 3 different missense mutations resulting in changes to 2 amino acids in all affected members of 11 HSN1 families. We found two mutations to be located in exon 5 (C133Y and C133W) and one mutation to be located in exon 6 of SPTLC1 (V144D). All families showing definite or probable linkage to chromosome 9 had mutations in these two exons. These mutations are associated with increased de novo glucosyl ceramide synthesis in lymphoblast cell lines in affected individuals. Increased de novo ceramide synthesis triggers apoptosis and is associated with massive cell death during neural tube closure, raising the possibility that neural degeneration in HSN1 is due to ceramide-induced apoptotic cell death.     

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('D?hle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.     

LMNA, encoding lamin A/C, is mutated in partial lipodystrophy

The lipodystrophies are a group of disorders characterized by the absence or reduction of subcutaneous adipose tissue. Partial lipodystrophy (PLD; MIM 151660) is an inherited condition in which a regional (trunk and limbs) loss of fat occurs during the peri-pubertal phase. Additionally, variable degrees of resistance to insulin action, together with a hyperlipidaemic state, may occur and simulate the metabolic features commonly associated with predisposition to atherosclerotic disease. The PLD locus has been mapped to chromosome 1q with no evidence of genetic heterogeneity. We, and others, have refined the location to a 5.3-cM interval between markers D1S305 and D1S1600 (refs 5, 6). Through a positional cloning approach we have identified five different missense mutations in LMNA among ten kindreds and three individuals with PLD. The protein product of LMNA is lamin A/C, which is a component of the nuclear envelope. Heterozygous mutations in LMNA have recently been identified in kindreds with the variant form of muscular dystrophy (MD) known as autosomal dominant Emery-Dreifuss MD (EDMD-AD; ref. 7) and dilated cardiomyopathy and conduction-system disease (CMD1A). As LMNA is ubiquitously expressed, the finding of site-specific amino acid substitutions in PLD, EDMD-AD and CMD1A reveals distinct functional domains of the lamin A/C protein required for the maintenance and integrity of different cell types.