Mutations in the SLC2A9 gene cause hyperuricosuria and hyperuricemia in the dog

Allantoin is the end product of purine catabolism in all mammals except humans, great apes, and one breed of dog, the Dalmatian. Humans and Dalmatian dogs produce uric acid during purine degradation, which leads to elevated levels of uric acid in blood and urine and can result in significant diseases in both species. The defect in Dalmatians results from inefficient transport of uric acid in both the liver and renal proximal tubules. Hyperuricosuria and hyperuricemia (huu) is a simple autosomal recessive trait for which all Dalmatian dogs are homozygous. Therefore, in order to map the locus, an interbreed backcross was used. Linkage mapping localized the huu trait to CFA03, which excluded the obvious urate transporter 1 gene, SLC22A12. Positional cloning placed the locus in a minimal interval of 2.5 Mb with a LOD score of 17.45. A critical interval of 333 kb containing only four genes was homozygous in all Dalmatians. Sequence and expression analyses of the SLC2A9 gene indicated three possible mutations, a missense mutation (G616T;C188F) and two promoter mutations that together appear to reduce the expression levels of one of the isoforms. The missense mutation is associated with hyperuricosuria in the Dalmatian, while the promoter SNPs occur in other unaffected breeds of dog. Verification of the causative nature of these changes was obtained when hyperuricosuric dogs from several other breeds were found to possess the same combination of mutations as found in the Dalmatian. The Dalmatian dog model of hyperuricosuria and hyperuricemia underscores the importance of SLC2A9 for uric acid transport in mammals.     

Mutations in SLC45A2 cause plumage color variation in chicken and Japanese quail

S*S (Silver), S*N (wild type/gold), and S*AL (sex-linked imperfect albinism) form a series of alleles at the S (Silver) locus on chicken (Gallus gallus) chromosome Z. Similarly, sex-linked imperfect albinism (AL*A) is the bottom recessive allele at the orthologous AL locus in Japanese quail (Coturnix japonica). The solute carrier family 45, member 2, protein (SLC45A2), previously denoted membrane-associated transporter protein (MATP), has an important role in vesicle sorting in the melanocytes. Here we report five SLC45A2 mutations. The 106delT mutation in the chicken S*AL allele results in a frameshift and a premature stop codon and the corresponding mRNA appears to be degraded by nonsense-mediated mRNA decay. A splice-site mutation in the Japanese quail AL*A allele causes in-frame skipping of exon 4. Two independent missense mutations (Tyr277Cys and Leu347Met) were associated with the Silver allele in chicken. The functional significance of the former mutation, associated only with Silver in White Leghorn, is unclear. Ala72Asp was associated with the cinnamon allele (AL*C) in the Japanese quail. The most interesting feature concerning the SLC45A2 variants documented in this study is the specific inhibition of expression of red pheomelanin in Silver chickens. This phenotypic effect cannot be explained on the basis of the current, incomplete, understanding of SLC45A2 function. It is an enigma why recessive null mutations at this locus cause an almost complete absence of both eumelanin and pheomelanin whereas some missense mutations are dominant and cause a specific inhibition of pheomelanin production.     

Familial pulmonary alveolar microlithiasis diagnosed by bronchoalveolar lavage. A case report

BACKGROUND: Familial alveolar microlithiasis is a rare lung disease. In this study we describe the cytologic features of this disease in bronchoalveolar lavage. CASE: A 10-year-old girl and her uncle, a 50-year-old man, had dyspnea and diffuse interstitial pattern on chest radiograph with no defined cause at a period of 10 years apart. Open lung biopsy in the girl and transbronchial lung biopsy plus bronchoalveolar lavage (BAL) in the man were per-formed to determine the diagnosis. In cyopen lung biopsy the diagnosis was alveolar microlithiasis. BAL revealed rehtypical microliths (calcospherites), and th transbronchial lung biopsy performed in the same patient failed to disclose superficially reset any significant pathology. In cytologic a smears, extracellular and intracellular concentrically layered purple-brown, round-to-oval microliths were clearly seen. Cyanophilic periodic acid-Schiff positive intracytoplasmic amorphous material was also frequently seen in alveolar macrophages. CONCLUSION: Familial alveolar microlithiasis is a rare interstitial lung disease that can be easily diagnosed by BAL. This procedure is a very useful tool in diagnosing and classifying some interstitial lung diseases.     

Mutations within the human GLYT2 (SLC6A5) gene associated with hyperekplexia

Hereditary hyperekplexia is a neuromotor disorder characterized by exaggerated startle reflexes and muscle stiffness in the neonate. The disease has been associated with mutations in the glycine receptor subunit genes GLRA1 and GLRB. Here, we describe mutations within the neuronal glycine transporter 2 gene (GLYT2, or SLC6A5, ) of hyperekplexia patients, whose symptoms cannot be attributed to glycine receptor mutations. One of the GLYT2 mutations identified causes truncation of the transporter protein and a complete loss of transport function. Our results are consistent with GLYT2 being a disease gene in human hyperekplexia.     

Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.     

Long-Term Cochlear Implant Outcomes in Children with GJB2 and SLC26A4 Mutations

Objectives

To investigate speech and language outcomes in children with cochlear implants (CIs) who had mutations in common deafness genes and to compare their performances with those without mutations.

Study Design

Prospective study.

Methods

Patients who received CIs before 18 years of age and had used CIs for more than 3 years were enrolled in this study. All patients underwent mutation screening of three common deafness genes: GJB2, SLC26A4 and the mitochondrial 12S rRNA gene. The outcomes with CIs were assessed at post-implant years 3 and 5 using the Categories of Auditory Performance (CAP) scale, Speech Intelligibility Rating (SIR) scale, speech perception tests and language skill tests.

Results

Forty-eight patients were found to have confirmative mutations in GJB2 or SLC26A4, and 123 without detected mutations were ascertained for comparison. Among children who received CIs before 3.5 years of age, patients with GJB2 or SLC26A4 mutations showed significantly higher CAP/SIR scores than those without mutations at post-implant year 3 (p = 0.001 for CAP; p = 0.004 for SIR) and year 5 (p = 0.035 for CAP; p = 0.038 for SIR). By contrast, among children who received CIs after age 3.5, no significant differences were noted in post-implant outcomes between patients with and without mutations (all p > 0.05).

Conclusion

GJB2 and SLC26A4 mutations are associated with good post-implant outcomes. However, their effects on CI outcomes may be modulated by the age at implantation: the association between mutations and CI outcomes is observed in young recipients who received CIs before age 3.5 years but not in older recipients.     

Iron deposition and increased alveolar septal capillary density in nonfibrotic lung tissue are associated with pulmonary hypertension in idiopathic pulmonary fibrosis

Background

Early diagnosis of pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) has potential prognostic and therapeutic implications but can be difficult due to the lack of specific clinical manifestations or accurate non-invasive tests. Histopathologic parameters correlating with PH in IPF are also not known. Remodeling of postcapillary pulmonary vessels has been reported in the nonfibrotic areas of explanted lungs from IPF patients. We hypothesized that iron deposition and increased alveolar capillaries, the findings often seen in postcapillary PH, might predict the presence of clinical PH, independent of the severity of fibrosis or ventilatory dysfunction in IPF patients. To test this hypothesis, we examined the association between these histologic parameters and the degree of PH, with consideration of the severity of disease in IPF.

Methods

Iron deposition and alveolar septal capillary density (ASCD) were evaluated on histologic sections with hematoxylin-eosin, iron, elastin and CD34 stainings. Percentage of predicted forced vital capacity (FVC%) was used for grading pulmonary function status. Fibrosis score assessed on high resolution computed tomography (HRCT) was used for evaluating overall degree of fibrosis in whole lungs. Right ventricular systolic pressure (RVSP) by transthoracic echocardiography was used for the estimation of PH. Univariate and multivariate regression analyses were performed.

Results

A cohort of 154 patients was studied who had the clinicopathological diagnosis of IPF with surgical lung biopsies or explants during the period of 1997 to 2006 at Mayo Clinic Rochester. In univariate analysis, RVSP in our IPF cases was associated with both iron deposition and ASCD (p < 0.001). In multivariate analysis with FVC% and HRCT fibrosis score included, iron deposition (p = 0.02), but not ASCD (p = 0.076), maintained statistically significant association with RVSP. FVC% was associated with RVSP on univariate analysis but not on multivariate analysis, while fibrosis score lacked any association with RVSP by either univariate or multivariate analyses.

Conclusions

Iron deposition and ASCD in non fibrotic lung tissue showed an association with RVSP, suggesting that these features are possible morphologic predictors of PH in IPF.     

SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (P(i)). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i)-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i) exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i)-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.     

CR2 units of CR2 complexes are possibly associated with nucleophilic agents through reactive covalent links

The human complement receptor type 2 (CR2/CD21), a transmembrane glycoprotein, associates with a variety of surface antigens and proteins in the cell membrane. We examined the possibilities that the CR2 units of CR2 complexes are associated through internal covalent links reactive with nucleophilic agents, e.g. H(2)O or methylamine, and that CR2-positive cells process anti-CR2 monoclonal antibodies (MoAbs). Data from immunoblotting and cytofluorimetry with CR2-binding site-specific MoAbs show that: (i) CR2-positive Raji cells release soluble CR2 isoforms into the medium when incubated in phosphate buffered saline; (ii) despite affecting the detection of one soluble CR2 isoform, methylamine treatment of soluble CR2 allows the detection of another of its isoforms; (iii) limited pre-treatment of cells with methylamine reveals a more heterogeneous CR2-positive cell population or enhances the detection of CR2; (iv) cell treatment with CR2-binding site-specific MoAbs enhances the detection of CR2 isoform(s). The data suggest that CR2 is shed mainly as a soluble CR2 complex, in which the CR2 units link covalently and react with nucleophilic agents. Raji cells may process bound fragments (145 kDa) that are recognised by and become bound by anti-CR2 MoAb.     

Mutations in Glucose Transporter 9 Gene SLC2A9 Cause Renal Hypouricemia

Renal hypouricemia is an inherited disorder characterized by impaired renal urate (uric acid) reabsorption and subsequent low serum urate levels, with severe complications such as exercise-induced acute renal failure and nephrolithiasis. We previously identified SLC22A12, also known as URAT1, as a causative gene of renal hypouricemia. However, hypouricemic patients without URAT1 mutations, as well as genome-wide association studies between urate and SLC2A9 (also called GLUT9), imply that GLUT9 could be another causative gene of renal hypouricemia. With a large human database, we identified two loss-of-function heterozygous mutations in GLUT9, which occur in the highly conserved “sugar transport proteins signatures 1/2.” Both mutations result in loss of positive charges, one of which is reported to be an important membrane topology determinant. The oocyte expression study revealed that both GLUT9 isoforms showed high urate transport activities, whereas the mutated GLUT9 isoforms markedly reduced them. Our findings, together with previous reports on GLUT9 localization, suggest that these GLUT9 mutations cause renal hypouricemia by their decreased urate reabsorption on both sides of the renal proximal tubules. These findings also enable us to propose a physiological model of the renal urate reabsorption in which GLUT9 regulates serum urate levels in humans and can be a promising therapeutic target for gout and related cardiovascular diseases.     

Mutations in the HFE, TFR2, and SLC40A1 genes in patients with hemochromatosis

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype.     

Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology

We have previously linked families with autosomal-dominant, late-onset parkinsonism to chromosome 12p11.2-q13.1 (PARK8). By high-resolution recombination mapping and candidate gene sequencing in 46 families, we have found six disease-segregating mutations (five missense and one putative splice site mutation) in a gene encoding a large, multifunctional protein, LRRK2 (leucine-rich repeat kinase 2). It belongs to the ROCO protein family and includes a protein kinase domain of the MAPKKK class and several other major functional domains. Within affected carriers of families A and D, six post mortem diagnoses reveal brainstem dopaminergic degeneration accompanied by strikingly diverse pathologies. These include abnormalities consistent with Lewy body Parkinson's disease, diffuse Lewy body disease, nigral degeneration without distinctive histopathology, and progressive supranuclear palsy-like pathology. Clinical diagnoses of Parkinsonism with dementia or amyotrophy or both, with their associated pathologies, are also noted. Hence, LRRK2 may be central to the pathogenesis of several major neurodegenerative disorders associated with parkinsonism.     

Mutations in EZH2 cause Weaver syndrome

We used trio-based whole-exome sequencing to analyze two families affected by Weaver syndrome, including one of the original families reported in 1974. Filtering of rare variants in the affected probands against the parental variants identified two different de novo mutations in the enhancer of zeste homolog 2 (EZH2). Sanger sequencing of EZH2 in a third classically-affected proband identified a third de novo mutation in this gene. These data show that mutations in EZH2 cause Weaver syndrome.     

AVPR1a and SLC6A4 gene polymorphisms are associated with creative dance performance

Dancing, which is integrally related to music, likely has its origins close to the birth of Homo sapiens, and throughout our history, dancing has been universally practiced in all societies. We hypothesized that there are differences among individuals in aptitude, propensity, and need for dancing that may partially be based on differences in common genetic polymorphisms. Identifying such differences may lead to an understanding of the neurobiological basis of one of mankind's most universal and appealing behavioral traits—dancing. In the current study, 85 current performing dancers and their parents were genotyped for the serotonin transporter (SLC6A4: promoter region HTTLPR and intron 2 VNTR) and the arginine vasopressin receptor 1a (AVPR1a: promoter microsatellites RS1 and RS3). We also genotyped 91 competitive athletes and a group of nondancers/nonathletes (n = 872 subjects from 414 families). Dancers scored higher on the Tellegen Absorption Scale, a questionnaire that correlates positively with spirituality and altered states of consciousness, as well as the Reward Dependence factor in Cloninger's Tridimensional Personality Questionnaire, a measure of need for social contact and openness to communication. Highly significant differences in AVPR1a haplotype frequencies (RS1 and RS3), especially when conditional on both SLC6A4 polymorphisms (HTTLPR and VNTR), were observed between dancers and athletes using the UNPHASED program package (Cocaphase: likelihood ratio test [LRS] = 89.23, p = 0.000044). Similar results were obtained when dancers were compared to nondancers/nonathletes (Cocaphase: LRS = 92.76, p = 0.000024). These results were confirmed using a robust family-based test (Tdtphase: LRS = 46.64, p = 0.010). Association was also observed between Tellegen Absorption Scale scores and AVPR1a (Qtdtphase: global chi-square = 26.53, p = 0.047), SLC6A4 haplotypes (Qtdtphase: chi-square = 2.363, p = 0.018), and AVPR1a conditional on SCL6A4 (Tdtphase: LRS = 250.44, p = 0.011). Similarly, significant association was observed between Tridimensional Personality Questionnaire Reward Dependence scores and AVPR1a RS1 (chi-square = 20.16, p = 0.01). Two-locus analysis (RS1 and RS3 conditional on HTTLPR and VNTR) was highly significant (LRS = 162.95, p = 0.001). Promoter repeat regions in the AVPR1a gene have been robustly demonstrated to play a role in molding a range of social behaviors in many vertebrates and, more recently, in humans. Additionally, serotonergic neurotransmission in some human studies appears to mediate human religious and spiritual experiences. We therefore hypothesize that the association between AVPR1a and SLC6A4 reflects the social communication, courtship, and spiritual facets of the dancing phenotype rather than other aspects of this complex phenotype, such as sensorimotor integration.     

Mutations in the NRG1 gene are associated with Hirschsprung disease

Hirschsprung disease (HSCR, congenital colon aganglionosis) is a relatively common complex genetic condition caused by abnormal development of the enteric nervous system (ENS). Through a recent genome-wide association study conducted on Chinese HSCR patients, we identified a new HSCR contributing locus, neuregulin 1 (NRG1; 8p12), a gene known to be involved in the development of the ENS. As genes in which disease-associated common variants are found are to be considered as candidates for the search of deleterious rare variants (RVs) in the coding sequences, we sequenced the NRG1 exons of 358 sporadic HSCR patients and 333 controls. We identified a total of 13 different heterozygous RVs including 8 non-synonymous (A28G, E134K, V266L, H347Y, P356L, V486M, A511T, P608A) and 3 synonymous amino acid substitutions (P24P, T169T, L483L), a frameshift (E239fsX10), and a c.503-4insT insertion. Functional analysis of the most conserved non-synonymous substitutions, H347Y and P356L, showed uneven intracellular distribution and aberrant expression of the mutant proteins. Except for T169T and V486M, all variants were exclusive to HSCR patients. Overall, there was a statistically significant over-representation of NRG1 RVs in HSCR patients (p?=?0.008). We show here that not only common, but also rare variants of the NRG1 gene contribute to HSCR. This strengthens the role of NRG1.     

Mutations in KIF11 cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.     

Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin

Low copper and ceruloplasmin in serum are the diagnostic hallmarks for Menkes disease, Wilson disease, and aceruloplasminemia. We report on five patients from four unrelated families with these biochemical findings who presented with a lethal autosomal-recessive syndrome of congenital cataracts, hearing loss, and severe developmental delay. Cerebral MRI showed pronounced cerebellar hypoplasia and hypomyelination. Homozygosity mapping was performed and displayed a region of commonality among three families at chromosome 3q25. Deep sequencing and conventional sequencing disclosed homozygous or compound heterozygous mutations for all affected subjects in SLC33A1 encoding a highly conserved acetylCoA transporter (AT-1) required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We also showed that AT-1 knockdown in HepG2 cells leads to reduced ceruloplasmin secretion, indicating that the low copper in serum is due to reduced ceruloplasmin levels and is not a sign of copper deficiency. The severity of the phenotype implies an essential role of AT-1 in proper posttranslational modification of numerous proteins, without which normal lens and brain development is interrupted. Furthermore, AT-1 defects are a new and important differential diagnosis in patients with low copper and ceruloplasmin in serum.     

Mutations in the BRWD3 gene cause X-linked mental retardation associated with macrocephaly

In the course of systematic screening of the X-chromosome coding sequences in 250 families with nonsyndromic X-linked mental retardation (XLMR), two families were identified with truncating mutations in BRWD3, a gene encoding a bromodomain and WD-repeat domain–containing protein. In both families, the mutation segregates with the phenotype in affected males. Affected males have macrocephaly with a prominent forehead, large cupped ears, and mild-to-moderate intellectual disability. No truncating variants were found in 520 control X chromosomes. BRWD3 is therefore a new gene implicated in the etiology of XLMR associated with macrocephaly and may cause disease by altering intracellular signaling pathways affecting cellular proliferation.