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1.  Coding and noncoding expression patterns associated with rare obesity-related disorders: Prader–Willi and Alström syndromes 
Advances in genomics and genetics  2015;2015(5):53-75.
Obesity is accompanied by hyperphagia in several classical genetic obesity-related syndromes that are rare, including Prader–Willi syndrome (PWS) and Alström syndrome (ALMS). We compared coding and noncoding gene expression in adult males with PWS, ALMS, and nonsyndromic obesity relative to nonobese males using readily available lymphoblastoid cells to identify disease-specific molecular patterns and disturbed mechanisms in obesity. We found 231 genes upregulated in ALMS compared with nonobese males, but no genes were found to be upregulated in obese or PWS males and 124 genes were downregulated in ALMS. The metallothionein gene (MT1X) was significantly downregulated in ALMS, in common with obese males. Only the complex SNRPN locus was disturbed (downregulated) in PWS along with several downregulated small nucleolar RNAs (snoRNAs) in the 15q11-q13 region (SNORD116, SNORD109B, SNORD109A, SNORD107). Eleven upregulated and ten downregulated snoRNAs targeting multiple genes impacting rRNA processing, developmental pathways, and associated diseases were found in ALMS. Fifty-two miRNAs associated with multiple, overlapping gene expression disturbances were upregulated in ALMS, and four were shared with obese males but not PWS males. For example, seven passenger strand microRNAs (miRNAs) (miR-93*, miR-373*, miR-29b-2*, miR-30c-1*, miR27a*, miR27b*, and miR-149*) were disturbed in association with six separate downregulated target genes (CD68, FAM102A, MXI1, MYO1D, TP53INP1, and ZRANB1). Cell cycle (eg, PPP3CA), transcription (eg, POLE2), and development may be impacted by upregulated genes in ALMS, while downregulated genes were found to be involved with metabolic processes (eg, FABP3), immune responses (eg, IL32), and cell signaling (eg, IL1B). The high number of gene and noncoding RNA disturbances in ALMS contrast with observations in PWS and males with nonsyndromic obesity and may reflect the progressing multiorgan pathology of the ALMS disease process.
doi:10.2147/AGG.S74598
PMCID: PMC4334166
hyperphagia; microarray analysis; gene; obesity; exon expression; miRNA expression
2.  Genetic modifier loci of mouse Mfrprd6 identified by quantitative trait locus analysis 
Experimental eye research  2013;118:30-35.
The identification of genes that modify pathological ocular phenotypes in mouse models may improve our understanding of disease mechanisms and lead to new treatment strategies. Here, we identify modifier loci affecting photoreceptor cell loss in homozygous Mfrprd6 mice, which exhibit a slowly progressive photoreceptor degeneration. A cohort of 63 F2 homozygous Mfrprd6 mice from a (B6.C3Ga-Mfrprd6/J × CAST/EiJ) F1 intercross exhibited a variable number of cell bodies in the retinal outer nuclear layer at 20 weeks of age. Mice were genotyped with a panel of single nucleotide polymorphism markers, and genotypes were correlated with phenotype by quantitative trait locus (QTL) analysis to map modifier loci. A genome-wide scan revealed a statistically significant, protective candidate locus on CAST/EiJ Chromosome 1 and suggestive modifier loci on Chromosomes 6 and 11. Multiple regression analysis of a three-QTL model indicated that the modifier loci on Chromosomes 1 and 6 together account for 26% of the observed phenotypic variation, while the modifier locus on Chromosome 11 explains only an additional 4%. Our findings indicate that the severity of the Mfrprd6 retinal degenerative phenotype in mice depends on the strain genetic background and that a significant modifier locus on CAST/EiJ Chromosome 1 protects against Mfrprd6-associated photoreceptor loss.
doi:10.1016/j.exer.2013.10.020
PMCID: PMC4153418  PMID: 24200520
eye disease; retinal degeneration; MFRP; QTL analysis; modifier genes
3.  An Allele of Microtubule-Associated Protein 1A (Mtap1a) Reduces Photoreceptor Degeneration in Tulp1 and Tub Mutant Mice 
The characterization of modifier genes can provide insights into disease pathways and identify novel therapeutic targets. This study identifies Mtap1a as a modifier gene of photoreceptor loss in Tulp1 and Tub mutant mice, which are models of retinal degeneration.
Purpose.
To identify genes that modify photoreceptor cell loss in the retinas of homozygous Tulp1tm1Pjn and Tubtub mice, which exhibit juvenile retinitis pigmentosa.
Methods.
Modifier loci were identified by genetic quantitative trait locus analysis. F2 Tulp1tm1Pjn/tm1Pjn mutant mice from a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross were genotyped with a panel of single nucleotide polymorphism markers and phenotyped by histology for photoreceptor nuclei remaining at 9 weeks of age. Genotype and phenotype data were correlated and examined with Pseudomarker 2.02 using 128 imputations to map modifier loci. Thresholds for the 63%, 10%, 5%, and 1% significance levels were obtained from 100 permutations. A significant, protective candidate modifier was identified by bioinformatic analysis and confirmed by crossing transgenic mice bearing a protective allele of this gene with Tulp1- and Tub-deficient mice.
Results.
A significant, protective modifier locus on chromosome 2 and a suggestive locus on chromosome 13 that increases photoreceptor loss were identified in a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross. The chromosome 2 locus mapped near Mtap1a, which encodes a protein associated with microtubule-based intracellular transport and synapse function. The protective Mtap1a129P2/OlaHsd allele was shown to reduce photoreceptor loss in both Tulp1tm1Pjn/tm1Pjn and Tubtub/tub mice.
Conclusions.
It was demonstrated that the gene Mtap1a, which modifies hearing loss in Tubtub/tub mice, also modifies retinal degeneration in Tubtub/tub and Tulp1tm1Pjn/tm1Pjn mice. These results suggest that functionally nonredundant members of the TULP family (TUB and TULP1) share a common functional interaction with MTAP1A.
doi:10.1167/iovs.11-8871
PMCID: PMC3339923  PMID: 22323461
4.  Meckelin Is Necessary for Photoreceptor Intraciliary Transport and Outer Segment Morphogenesis 
This study revealed a role for meckelin in the intraciliary trafficking of phototransduction molecules and in the elongation and maintenance of the photoreceptor outer segments.
Purpose.
Cilia, complex structures found ubiquitously in most vertebrate cells, serve a variety of functions ranging from cell and fluid movement, cell signaling, tissue homeostasis, to sensory perception. Meckelin is a component of ciliary and cell membranes and is encoded by Tmem67 (Mks3). In this study, the retinal morphology and ciliary function in a mouse model for Meckel Syndrome Type 3 (MKS3) throughout the course of photoreceptor development was examined.
Methods.
To study the effects of a disruption in the Mks3 gene on the retina, the authors introduced a functional allele of Pde6b into B6C3Fe a/a-bpck/J mice and evaluated their retinas by ophthalmoscopic, histologic, and ultrastructural examination. In addition, immunofluorescence microscopy was used to assess protein trafficking through the connecting cilium and to examine the localization of ciliary and synaptic proteins in Tmem67bpck mice and controls.
Results.
Photoreceptors degenerate early and rapidly in bpck/bpck mutant mice. In addition, phototransduction proteins, such as rhodopsin, arrestin, and transducin, are mislocalized. Ultrastructural examination of photoreceptors reveal morphologically intact connecting cilia but dysmorphic and misoriented outer segment (OS) discs, at the earliest time point examined.
Conclusions.
These findings underscore the important role for meckelin in intraciliary transport of phototransduction molecules and their effects on subsequent OS morphogenesis and maintenance.
doi:10.1167/iovs.11-8766
PMCID: PMC3317434  PMID: 22247471
6.  Gene Profiling of Postnatal Mfrprd6 Mutant Eyes Reveals Differential Accumulation of Prss56, Visual Cycle and Phototransduction mRNAs 
PLoS ONE  2014;9(10):e110299.
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.
doi:10.1371/journal.pone.0110299
PMCID: PMC4214712  PMID: 25357075
7.  GLUT4 Defects in Adipose Tissue Are Early Signs of Metabolic Alterations in Alms1GT/GT, a Mouse Model for Obesity and Insulin Resistance 
PLoS ONE  2014;9(10):e109540.
Dysregulation of signaling pathways in adipose tissue leading to insulin resistance can contribute to the development of obesity-related metabolic disorders. Alström Syndrome, a recessive ciliopathy, caused by mutations in ALMS1, is characterized by progressive metabolic alterations such as childhood obesity, hyperinsulinemia, and type 2 diabetes. Here we investigated the role of Alms1 disruption in AT expansion and insulin responsiveness in a murine model for Alström Syndrome. A gene trap insertion in Alms1 on the insulin sensitive C57BL6/Ei genetic background leads to early hyperinsulinemia and a progressive increase in body weight. At 6 weeks of age, before the onset of the metabolic disease, the mutant mice had enlarged fat depots with hypertrophic adipocytes, but without signs of inflammation. Expression of lipogenic enzymes was increased. Pre-adipocytes isolated from mutant animals demonstrated normal adipogenic differentiation but gave rise to mature adipocytes with reduced insulin-stimulated glucose uptake. Assessment of whole body glucose homeostasis revealed glucose intolerance. Insulin stimulation resulted in proper AKT phosphorylation in adipose tissue. However, the total amount of glucose transporter 4 (SLC4A2) and its translocation to the plasma membrane were reduced in mutant adipose depots compared to wildtype littermates. Alterations in insulin stimulated trafficking of glucose transporter 4 are an early sign of metabolic dysfunction in Alström mutant mice, providing a possible explanation for the reduced glucose uptake and the compensatory hyperinsulinemia. The metabolic signaling deficits either reside downstream or are independent of AKT activation and suggest a role for ALMS1 in GLUT4 trafficking. Alström mutant mice represent an interesting model for the development of metabolic disease in which adipose tissue with a reduced glucose uptake can expand by de novo lipogenesis to an obese state.
doi:10.1371/journal.pone.0109540
PMCID: PMC4192353  PMID: 25299671
8.  Degradation of IRS1 leads to impaired glucose uptake in adipose tissue of the type 2 diabetes mouse model TALLYHO/Jng 
The Journal of endocrinology  2009;203(1):65-74.
The TALLYHO/Jng (TH) mouse strain is a polygenic model for type 2 diabetes characterized by moderate obesity, impaired glucose tolerance and uptake, insulin resistance, and hyperinsulinemia. The goal of this study was to elucidate the molecular mechanisms responsible for the reduced glucose uptake and insulin resistance in the adipose tissue of this model.
The translocation and localization of GLUT4 to the adipocyte plasma membrane were impaired in TH mice compared to control C57BL6/J (B6) mice. These defects were associated with decreased GLUT4 protein, reduced PI3 kinase activity and alterations in the phosphorylation status of IRS1. Activation of JNK 1/2, which can phosphorylate IRS1 on Ser307, was significantly higher in TH mice compared to B6 controls. IRS1 protein but not mRNA levels were found to be lower in TH mice than controls. Immunoprecipitation with anti-ubiquitin and western blot analysis of IRS1 protein revealed increased total IRS1 ubiquitination in adipose tissue of TH mice. SOCS1, known to promote IRS1 ubiquitination and subsequent degradation, was found at significantly higher levels in TH mice compared to B6. Immunohistochemistry showed that IRS1 co-localized with the 20S proteasome in proteasomal structures in TH adipocytes, supporting the notion that IRS1 is actively degraded.
Our findings suggest that increased IRS1 degradation and subsequent impaired GLUT4 mobilization play a role in the reduced glucose uptake in insulin resistant TH mice. Since low IRS1 levels are often observed in human type 2 diabetes, the TH mouse is an attractive model to investigate mechanisms of insulin resistance and explore new treatments.
doi:10.1677/JOE-09-0026
PMCID: PMC2853731  PMID: 19587264
IRS1 degradation; adipose tissue; type 2 diabetes; mice
9.  A new mouse model of metabolic syndrome and associated complications 
The Journal of endocrinology  2009;202(1):17-28.
Metabolic Syndrome (MS) encompasses a clustering of risk factors for cardiovascular disease, including obesity, insulin resistance, and dyslipidemia. We characterized a new mouse model carrying a dominant mutation, C57BL/6J-Nmf15/+ (B6-Nmf15/+), which develops additional complications of MS such as adipose tissue inflammation and cardiomyopathy.
A backcross was used to genetically map the Nmf15 locus. Mice were examined in the CLAMS™ animal monitoring system, and dual energy X-ray absorptiometry and blood chemistry analyses were performed. Hypothalamic LepR, SOCS1 and STAT3 phosphorylation were examined. Cardiac function was assessed by Echo- and Electro Cardiography. Adipose tissue inflammation was characterized by in situ hybridization and measurement of Jun kinase activity.
The Nmf15 locus mapped to distal mouse chromosome 5 with a LOD score of 13.8. Nmf15 mice developed obesity by 12 weeks of age. Plasma leptin levels were significantly elevated in pre-obese Nmf15 mice at 8 weeks of age and an attenuated STAT3 phosphorylation in the hypothalamus suggests a primary leptin resistance. Adipose tissue from Nmf15 mice showed a remarkable degree of inflammation and macrophage infiltration as indicated by expression of the F4/80 marker and increased phosphorylation of JNK1/2. Lipidosis was observed in tubular epithelial cells and glomeruli of the kidney. Nmf15 mice demonstrate both histological and pathophysiological evidence of cardiomyopathy.
The Nmf15 mouse model provides a new entry point into pathways mediating leptin resistance and obesity. It is one of few models that combine many aspects of metabolic syndrome and can be useful for testing new therapeutic approaches for combating obesity complications, particularly cardiomyopathy.
doi:10.1677/JOE-09-0079
PMCID: PMC2853727  PMID: 19398498
mouse model; hyperleptinemic obesity; adipose inflammation; renal lipidosis; cardiomyopathy; metabolic syndrome
10.  Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes 
Nature communications  2014;5:3416.
Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.
doi:10.1038/ncomms4416
PMCID: PMC3992616  PMID: 24595103
11.  Extreme clinical variability of dilated cardiomyopathy in two siblings with Alström syndrome 
Pediatric cardiology  2012;34(2):455-458.
Alström syndrome (ALMS) is a rare autosomal recessive disorder caused by mutations in the ALMS1 gene. We report on two brothers, 2 and 3 years of age, diagnosed with Alström syndrome who initially presented in infancy with severe dilated cardiomyopathy during febrile respiratory infection. The disease course in the two siblings was marked by significant intra-familial variability. While cardiomyopathy in the older sibling has mainly resolved allowing for the discontinuation of medical therapy, heart function in the younger sibling continues to deteriorate despite maximal drug support with furosemide, carvedilol, captopril and aldospirone. Genetic analysis revealed homozygous mutations, c.8008C>T (R2670X), in ALMS1 resulting in premature protein truncation. This report further emphasizes the exceptional intra-familial variability of ALMS, mainly in the natural course of cardiac disease.
doi:10.1007/s00246-012-0296-6
PMCID: PMC3779600  PMID: 22447358
Alström syndrome; dilated cardiomyopathy; autosomal recessive; ALMS1 gene
12.  Differences in the clinical spectrum of two adolescent male patients with Alström syndrome 
Clinical dysmorphology  2013;22(1):7-12.
Alström syndrome is a rare disorder typified by early childhood obesity, neurosensory deficits, cardiomyopathy, progressive renal and hepatic dysfunction, and endocrinological features such as severe insulin resistance, type 2 diabetes, hyperlipidemia, and hypogonadism. Widespread fibrosis leads to multiple organ failure. Mutations in ALMS1 cause Alström’s syndrome. Two age-matched, unrelated adolescent males of Serbian descent with Alström syndrome underwent an extensive workup of blood chemistries, and ophthalmological, audiological, and genetic evaluations. Although both showed typical features of Alström syndrome in childhood, several differences were observed that have not been reported previously. Patient 1 was first studied at the age of 13 years for multisystemic disease and re-evaluated at the age of 15.5 years. Patient 2 is a 15-year-old boy who presented at birth with epilepsy and psychomotor developmental delay and generalized tonic–clonic seizures with severe cognitive impairment, features not documented previously in this syndrome. Sequencing analysis indicated two novel ALMS1 mutations in exon 8: p.E1055GfsX4 and p.T1386NfsX15. Metabolic and physiological similarities were observed in both patients, including severe insulin resistance, and truncal obesity with fat loss suggestive of partial lipodystrophy, supporting evidence for a role for ALMS1 in adipose tissue function. The unusual phenotypes of clonic–tonic seizures and severe cognitive abnormalities and lipodystrophy-like adiposity pattern have not been documented previously in Alström syndrome and may be an under-reported abnormality.
doi:10.1097/MCD.0b013e32835b9017
PMCID: PMC3619948  PMID: 23188138
ALMS1; Alström syndrome; hypogonadotropic hypogonadism; partial lipodystrophy; tonic–clonic epilepsy
13.  Novel Alu Retrotransposon Insertion Leading to Alström Syndrome 
Human Genetics  2011;131(3):407-413.
Alström Syndrome is a clinically complex disorder characterized by childhood retinal degeneration leading to blindness, sensorineural hearing loss, obesity, type 2 diabetes mellitus, cardiomyopathy, systemic fibrosis, and pulmonary, hepatic, and renal failure. Alström Syndrome is caused by recessively inherited mutations in the ALMS1 gene, which codes for a putative ciliary protein. Alström Syndrome is characterized by extensive allelic heterogeneity, however founder effects have been observed in some populations. To date, more than 100 causative ALMS1 mutations have been identified, mostly frameshift and nonsense alterations resulting in termination signals in ALMS1. Here we report a complex Turkish kindred in which sequence analysis uncovered an insertion of a novel 333 basepair Alu Ya5 SINE retrotransposon in the ALMS1 coding sequence, a previously unrecognized mechanism underlying mutations causing Alström Syndrome. It is extraordinarily rare to encounter the insertion of an Alu retrotransposon in the coding sequence of a gene. The high frequency of the mutant ALMS1 allele in this isolated population suggests that this recent retrotransposition event spread quickly, and may be used as a model to study the population dynamics of deleterious alleles in isolated communities.
doi:10.1007/s00439-011-1083-9
PMCID: PMC3264847  PMID: 21877133
Alström Syndrome; ALMS1; Alu Ya5; Insertion Mutation; Short Interspersed Nuclear Elements (SINE)
14.  Brain involvement in Alström syndrome 
Background
Alström Syndrome (AS) is a rare ciliopathy characterized by cone–rod retinal dystrophy, sensorineural hearing loss, obesity, type 2 diabetes mellitus and cardiomyopathy. Most patients do not present with neurological issues and demonstrate normal intelligence, although delayed psychomotor development and psychiatric disorders have been reported. To date, brain Magnetic Resonance Imaging (MRI) abnormalities in AS have not been explored.
Methods
We investigated structural brain changes in 12 genetically proven AS patients (mean-age 22 years; range: 6–45, 6 females) and 19 matched healthy and positive controls (mean-age 23 years; range: 6–43; 12 females) using conventional MRI, Voxel-Based Morphometry (VBM) and Diffusion Tensor Imaging (DTI).
Results
6/12 AS patients presented with brain abnormalities such as ventricular enlargement (4/12), periventricular white matter abnormalities (3/12) and lacune-like lesions (1/12); all patients older than 30 years had vascular-like lesions. VBM detected grey and white matter volume reduction in AS patients, especially in the posterior regions. DTI revealed significant fractional anisotropy decrease and radial diffusivity increase in the supratentorial white matter, also diffusely involving those regions that appeared normal on conventional imaging. On the contrary, axial and mean diffusivity did not differ from controls except in the fornix.
Conclusions
Brain involvement in Alström syndrome is not uncommon. Early vascular-like lesions, gray and white matter atrophy, mostly involving the posterior regions, and diffuse supratentorial white matter derangement suggest a role of cilia in endothelial cell and oligodendrocyte function.
doi:10.1186/1750-1172-8-24
PMCID: PMC3584911  PMID: 23406482
Alström syndrome; MRI; DTI; VBM
15.  The Alström Syndrome Protein, ALMS1, Interacts with α-Actinin and Components of the Endosome Recycling Pathway 
PLoS ONE  2012;7(5):e37925.
Alström syndrome (ALMS) is a progressive multi-systemic disorder characterized by cone-rod dystrophy, sensorineural hearing loss, childhood obesity, insulin resistance and cardiac, renal, and hepatic dysfunction. The gene responsible for Alström syndrome, ALMS1, is ubiquitously expressed and has multiple splice variants. The protein encoded by this gene has been implicated in ciliary function, cell cycle control, and intracellular transport. To gain better insight into the pathways through which ALMS1 functions, we carried out a yeast two hybrid (Y2H) screen in several mouse tissue libraries to identify ALMS1 interacting partners. The majority of proteins found to interact with the murine carboxy-terminal end (19/32) of ALMS1 were α-actinin isoforms. Interestingly, several of the identified ALMS1 interacting partners (α-actinin 1, α-actinin 4, myosin Vb, rad50 interacting 1 and huntingtin associated protein1A) have been previously associated with endosome recycling and/or centrosome function. We examined dermal fibroblasts from human subjects bearing a disruption in ALMS1 for defects in the endocytic pathway. Fibroblasts from these patients had a lower uptake of transferrin and reduced clearance of transferrin compared to controls. Antibodies directed against ALMS1 N- and C-terminal epitopes label centrosomes and endosomal structures at the cleavage furrow of dividing MDCK cells, respectively, suggesting isoform-specific cellular functions. Our results suggest a role for ALMS1 variants in the recycling endosome pathway and give us new insights into the pathogenesis of a subset of clinical phenotypes associated with ALMS.
doi:10.1371/journal.pone.0037925
PMCID: PMC3365098  PMID: 22693585
16.  Arrayed primer extension technology simplifies mutation detection in Bardet–Biedl and Alström syndrome 
Bardet–Biedl syndrome (BBS; OMIM no. 209 900) and Alström syndrome (ALMS; OMIM no. 203 800) are rare, multisystem genetic disorders showing both a highly variable phenotype and considerable phenotypic overlap; they are included in the emerging group of diseases called ciliopathies. The genetic heterogeneity of BBS with 14 causal genes described to date, serves to further complicate mutational analysis. The development of the BBS–ALMS array which detects known mutations in these genes has allowed us to detect at least one mutation in 40.5% of BBS families and in 26.7% of ALMS families validating this as an efficient and cost-effective first pass screening modality. Furthermore, using this method, we found two BBS families segregating three BBS alleles further supporting oligogenicity or modifier roles for additional mutations. We did not observe more than two mutations in any ALMS family.
doi:10.1038/ejhg.2010.207
PMCID: PMC3060323  PMID: 21157496
Bardet–Biedl syndrome; BBS; Alström syndrome; ALMS1; arrayed primer extension; mutation analysis
17.  NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development 
Human Molecular Genetics  2010;20(3):482-496.
Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4nmf192/nmf192 mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4nmf192/nmf192 mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4nmf192/nmf192 male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4nmf192/nmf192 mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.
doi:10.1093/hmg/ddq494
PMCID: PMC3016909  PMID: 21078623
18.  Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss 
Human molecular genetics  1999;8(9):1761-1767.
Quantitative trait locus (QTL) analysis of genetic crosses has proven to be a useful tool for identifying loci associated with specific phenotypes and for dissecting genetic components of complex traits. Inclusion of a mutation that interacts epistatically with QTLs in genetic crosses is a unique and potentially powerful method of revealing the function of novel genes and pathways. Although we know that a mutation within the novel tub gene leads to obesity and cochlear and retinal degeneration, the biological function of the gene and the mechanism by which it induces its phenotypes are not known. In the current study, a QTL analysis for auditory brainstem response (ABR) thresholds, which indicates hearing ability, was performed in tubby mice from F2 intercrosses between C57BL/6J-tub/tub and AKR/J-+/+ F1, hybrids (AKR intercross) and between C57BL/6J-tub/tub and CAST/Ei.B6-tub/tub F1 hybrids (CAST intercross). A major QTL, designated as modifier of tubby hearing1 (moth1), was identified on chromosome 2 with a LOD score of 33.4 (P <; 10−33) in the AKR intercross (181 mice) and of 6.0 (P <; 10−6) in the CAST intercross (46 mice). This QTL is responsible for 57 and 43% of ABR threshold variance, respectively, in each strain combination. In addition, a C57BL/6J congenic line carrying a 129/Ola segment encompassing the described QTL region when made homozygous for tubby also exhibits normal hearing ability. We hypothesize that C57BL/6J carries a recessive mutation of the moth1 gene which interacts with the tub mutation to cause hearing loss in tub/tub mice. A moth1 allele from either AKR/J, CAST/Ei or 129/Ola is sufficient to protect C57BL/6J-tub/tub mice from hearing loss.
PMCID: PMC3249642  PMID: 10441341
19.  An ENU-Induced Mutation in the Mertk Gene (Mertknmf12) Leads to a Slow Form of Retinal Degeneration 
This study illustrates the discovery of a MERTK model with a slowly progressive form of retinal degeneration. These mice also demonstrate protective accumulation of TNF in the retina.
Purpose.
To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration.
Methods.
Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2.
Results.
Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertknmf12. We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertknmf12 homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertknmf12 homozygotes. Mertknmf12 homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.2 B6.129P2-Ltb/Tnf/Ltatm1Dvk/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc−/− mice. Surprisingly, mice homozygous for both the Mertknmf12 and the Ltb/Tnf/Ltatm1Dvk allele (Tnfabc−/−) demonstrated an increase in the rate of retinal degeneration.
Conclusions.
These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertknmf12 homozygotes from cell death.
doi:10.1167/iovs.10-7077
PMCID: PMC3175976  PMID: 21436282
20.  Alström Syndrome: Genetics and Clinical Overview 
Current Genomics  2011;12(3):225-235.
Alström syndrome is a rare autosomal recessive genetic disorder characterized by cone-rod dystrophy, hearing loss, childhood truncal obesity, insulin resistance and hyperinsulinemia, type 2 diabetes, hypertriglyceridemia, short stature in adulthood, cardiomyopathy, and progressive pulmonary, hepatic, and renal dysfunction. Symptoms first appear in infancy and progressive development of multi-organ pathology leads to a reduced life expectancy. Variability in age of onset and severity of clinical symptoms, even within families, is likely due to genetic background.
Alström syndrome is caused by mutations in ALMS1, a large gene comprised of 23 exons and coding for a protein of 4,169 amino acids. In general, ALMS1 gene defects include insertions, deletions, and nonsense mutations leading to protein truncations and found primarily in exons 8, 10 and 16. Multiple alternate splice forms exist. ALMS1 protein is found in centrosomes, basal bodies, and cytosol of all tissues affected by the disease. The identification of ALMS1 as a ciliary protein explains the range of observed phenotypes and their similarity to those of other ciliopathies such as Bardet-Biedl syndrome.
Studies involving murine and cellular models of Alström syndrome have provided insight into the pathogenic mechanisms underlying obesity and type 2 diabetes, and other clinical problems. Ultimately, research into the pathogenesis of Alström syndrome should lead to better management and treatments for individuals, and have potentially important ramifications for other rare ciliopathies, as well as more common causes of obesity and diabetes, and other conditions common in the general population.
doi:10.2174/138920211795677912
PMCID: PMC3137007  PMID: 22043170
ALMS1; Alström syndrome; ciliopathy; truncal obesity.
21.  Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice 
The Journal of Clinical Investigation  2011;121(6):2169-2180.
The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.
doi:10.1172/JCI45627
PMCID: PMC3104757  PMID: 21606596
22.  ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis 
PLoS ONE  2011;6(4):e19081.
Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions.
doi:10.1371/journal.pone.0019081
PMCID: PMC3082548  PMID: 21541333
23.  Photoreceptor Degeneration, Azoospermia, Leukoencephalopathy, and Abnormal RPE Cell Function in Mice Expressing an Early Stop Mutation in CLCN2 
This study characterizes a chemically induced mutation leading to an early stop codon in CLCN2 that causes photoreceptor degeneration, leukoencephalopathy, and azoospermia. Loss of one functional Clcn2 allele significantly reduced the electroretinogram light peak response, suggesting that this chloride channel is necessary for the generation of this response.
Purpose.
To determine the molecular basis and the pathologic consequences of a chemically induced mutation in a mouse model of photoreceptor degeneration, nmf240.
Methods.
Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened by indirect ophthalmoscopy for abnormal fundi. A chromosomal position for the recessive nmf240 mutation was determined by a genome-wide linkage analysis by use of simple sequence length polymorphic markers in an F2 intercross. The critical region was refined, and candidate genes were screened by direct sequencing. The nmf240 phenotype was characterized by histologic analysis of the retina, brain, and male reproductive organs and by electroretinogram (ERG)-based studies of the retina and retinal pigment epithelium (RPE).
Results.
Clinically, homozygous nmf240 mutants exhibit a grainy retina that progresses to panretinal patches of depigmentation. The mutation was localized to a region on chromosome 16 containing Clcn2, a gene associated with retinal degeneration. Sequencing identified a missense C-T mutation at nucleotide 1063 in Clcn2 that converts a glutamine to a stop codon. Mice homozygous for the Clcn2nmf240 mutation experience a severe loss of photoreceptor cells at 14 days of age that is preceded by an elongation of RPE apical microvilli. Homozygous mutants also experience leukoencephalopathy in multiple brain areas and male sterility. Despite a normal retinal histology in nmf240 heterozygotes, the ERG light peak, generated by the RPE, is reduced.
Conclusions.
The nmf240 phenotype closely resembles that reported for Clcn2 knockout mice. The observation that heterozygous nmf240 mice present with a reduced ERG light peak component suggests that CLCN2 is necessary for the generation of this response component.
doi:10.1167/iovs.09-4887
PMCID: PMC2891478  PMID: 20071672
24.  RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis 
Human Molecular Genetics  2009;18(22):4329-4339.
The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1tm1Tili mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1nmf247 that is phenotypically distinct from Rpgrip1tm1Tili mice. Photoreceptor degeneration in homozygous Rpgrip1nmf247 mice is earlier in onset and more severe when compared with Rpgrip1tm1Tili mice. Also, ultrastructural studies reveal that whereas Rpgrip1nmf247 mutants have a normal structure and number of connecting cilia, unlike Rpgrip1tm1Tili mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1nmf247 mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.
doi:10.1093/hmg/ddp385
PMCID: PMC2766293  PMID: 19679561
25.  Mouse Model Resources for Vision Research 
Journal of Ophthalmology  2010;2011:391384.
The need for mouse models, with their well-developed genetics and similarity to human physiology and anatomy, is clear and their central role in furthering our understanding of human disease is readily apparent in the literature. Mice carrying mutations that alter developmental pathways or cellular function provide model systems for analyzing defects in comparable human disorders and for testing therapeutic strategies. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Two programs, the Eye Mutant Resource and the Translational Vision Research Models, focused on providing such models to the vision research community are described herein. Over 100 mutant lines from the Eye Mutant Resource and 60 mutant lines from the Translational Vision Research Models have been developed. The ocular diseases of the mutant lines include a wide range of phenotypes, including cataracts, retinal dysplasia and degeneration, and abnormal blood vessel formation. The mutations in disease genes have been mapped and in some cases identified by direct sequencing. Here, we report 3 novel alleles of Crxtvrm65, Rp1tvrm64, and Rpe65tvrm148 as successful examples of the TVRM program, that closely resemble previously reported knockout models.
doi:10.1155/2011/391384
PMCID: PMC2968714  PMID: 21052544

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