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1.  Spectrum of PEX1 and PEX6 variants in Heimler syndrome 
Heimler syndrome (HS) consists of recessively inherited sensorineural hearing loss, amelogenesis imperfecta (AI) and nail abnormalities, with or without visual defects. Recently HS was shown to result from hypomorphic mutations in PEX1 or PEX6, both previously implicated in Zellweger Syndrome Spectrum Disorders (ZSSD). ZSSD are a group of conditions consisting of craniofacial and neurological abnormalities, sensory defects and multi-organ dysfunction. The finding of HS-causing mutations in PEX1 and PEX6 shows that HS represents the mild end of the ZSSD spectrum, though these conditions were previously thought distinct nosological entities. Here, we present six further HS families, five with PEX6 variants and one with PEX1 variants, and show the patterns of Pex1, Pex14 and Pex6 immunoreactivity in the mouse retina. While Ratbi et al. found more HS-causing mutations in PEX1 than PEX6, as is the case for ZSSD, in this cohort PEX6 variants predominate, suggesting both genes play a significant role in HS. The PEX6 variant c.1802G>A, p.(R601Q), reported previously in compound heterozygous state in one HS and three ZSSD cases, was found in compound heterozygous state in three HS families. Haplotype analysis suggests a common founder variant. All families segregated at least one missense variant, consistent with the hypothesis that HS results from genotypes including milder hypomorphic alleles. The clinical overlap of HS with the more common Usher syndrome and lack of peroxisomal abnormalities on plasma screening suggest that HS may be under-diagnosed. Recognition of AI is key to the accurate diagnosis of HS.
doi:10.1038/ejhg.2016.62
PMCID: PMC5026821  PMID: 27302843
Heimler syndrome; PEX1; PEX6; peroxisomal biogenesis disorders; amelogenesis imperfecta; retinal dystrophy; hearing loss; peroxisomes; Zellweger syndrome; Infantile Refsum Disease; neonatal adrenoleukodystrophy
2.  Spectrum of PEX1 and PEX6 variants in Heimler syndrome 
European Journal of Human Genetics  2016;24(11):1565-1571.
Heimler syndrome (HS) consists of recessively inherited sensorineural hearing loss, amelogenesis imperfecta (AI) and nail abnormalities, with or without visual defects. Recently HS was shown to result from hypomorphic mutations in PEX1 or PEX6, both previously implicated in Zellweger Syndrome Spectrum Disorders (ZSSD). ZSSD are a group of conditions consisting of craniofacial and neurological abnormalities, sensory defects and multi-organ dysfunction. The finding of HS-causing mutations in PEX1 and PEX6 shows that HS represents the mild end of the ZSSD spectrum, though these conditions were previously thought to be distinct nosological entities. Here, we present six further HS families, five with PEX6 variants and one with PEX1 variants, and show the patterns of Pex1, Pex14 and Pex6 immunoreactivity in the mouse retina. While Ratbi et al. found more HS-causing mutations in PEX1 than in PEX6, as is the case for ZSSD, in this cohort PEX6 variants predominate, suggesting both genes play a significant role in HS. The PEX6 variant c.1802G>A, p.(R601Q), reported previously in compound heterozygous state in one HS and three ZSSD cases, was found in compound heterozygous state in three HS families. Haplotype analysis suggests a common founder variant. All families segregated at least one missense variant, consistent with the hypothesis that HS results from genotypes including milder hypomorphic alleles. The clinical overlap of HS with the more common Usher syndrome and lack of peroxisomal abnormalities on plasma screening suggest that HS may be under-diagnosed. Recognition of AI is key to the accurate diagnosis of HS.
doi:10.1038/ejhg.2016.62
PMCID: PMC5026821  PMID: 27302843
3.  Whole Exome Sequencing Reveals Mutations in Known Retinal Disease Genes in 33 out of 68 Israeli Families with Inherited Retinopathies 
Scientific Reports  2015;5:13187.
Whole exome sequencing (WES) is a powerful technique for identifying sequence changes in the human genome. The goal of this study was to delineate the genetic defects in patients with inherited retinal diseases (IRDs) using WES. WES was performed on 90 patient DNA samples from 68 families and 226 known genes for IRDs were analyzed. Sanger sequencing was used to validate potential pathogenic variants that were also subjected to segregation analysis in families. Thirty-three causative mutations (19 novel and 14 known) in 25 genes were identified in 33 of the 68 families. The vast majority of mutations (30 out of 33) have not been reported in the Israeli and the Palestinian populations. Nine out of the 33 mutations were detected in additional families from the same ethnic population, suggesting a founder effect. In two families, identified phenotypes were different from the previously reported clinical findings associated with the causative gene. This is the largest genetic analysis of IRDs in the Israeli and Palestinian populations to date. We also demonstrate that WES is a powerful tool for rapid analysis of known disease genes in large patient cohorts.
doi:10.1038/srep13187
PMCID: PMC4549705  PMID: 26306921
4.  Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) 
Human Molecular Genetics  2015;24(13):3742-3751.
Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.
doi:10.1093/hmg/ddv118
PMCID: PMC4459392  PMID: 25859010
5.  An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy 
Molecular Vision  2015;21:1295-1306.
Purpose
To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family.
Methods
Patients underwent a detailed ophthalmic evaluation, including eye examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potential (VEP). Genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing.
Results
The affected family members are three siblings who have various degrees of progressive visual deterioration, glare, color vision abnormalities, and night vision difficulties. Visual field tests revealed central scotomas of different extension. Cone and rod ERG responses were reduced, with cones more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice sites. However, in intron 21, proximate to the intron–exon junction, we observed a homozygous 10 bp deletion between positions −26 and −17 (c.2281–26_-17del). The deletion was linked to a known SNP, c.2281–6C>G. The deletion cosegregated with the disease in the family, and was not detected in public databases or in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a recognition site for the SRSF2 splicing factor is located within the deleted sequence. The in vitro splicing assay demonstrated that c.2281–26_-17del leads to complete exon 22 skipping.
Conclusions
A novel and unique intronic mutation of PROM1, underlying autosomal recessive CRD in a consanguineous Israeli family, was found. This report expands the spectrum of pathogenic mutations of PROM1 and further demonstrates the importance of intronic mutations.
PMCID: PMC4676936  PMID: 26702251
6.  Genetic heterogeneity and consanguinity lead to a “double hit”: Homozygous mutations of MYO7A and PDE6B in a patient with retinitis pigmentosa 
Molecular Vision  2013;19:1565-1571.
Purpose
Retinitis pigmentosa (RP), the most genetically heterogeneous disorder in humans, actually represents a group of pigmentary retinopathies characterized by night blindness followed by visual-field loss. RP can appear as either syndromic or nonsyndromic. One of the most common forms of syndromic RP is Usher syndrome, characterized by the combination of RP, hearing loss, and vestibular dysfunction.
Methods
The underlying cause of the appearance of syndromic and nonsyndromic RP in three siblings from a consanguineous Israeli Muslim Arab family was studied with whole-genome homozygosity mapping followed by whole exome sequencing.
Results
The family was found to segregate novel mutations of two different genes: myosin VIIA (MYO7A), which causes type 1 Usher syndrome, and phosphodiesterase 6B, cyclic guanosine monophosphate-specific, rod, beta (PDE6B), which causes nonsyndromic RP. One affected child was homozygous for both mutations. Since the retinal phenotype seen in this patient results from overlapping pathologies, one might expect to find severe retinal degeneration. Indeed, he was diagnosed with RP based on an abnormal electroretinogram (ERG) at a young age (9 months). However, this early diagnosis may be biased, as two of his older siblings had already been diagnosed, leading to increased awareness. At the age of 32 months, he had relatively good vision with normal visual fields. Further testing of visual function and structure at different ages in the three siblings is needed to determine whether the two RP-causing genes mutated in this youngest sibling confer increased disease severity.
Conclusions
This report further supports the genetic heterogeneity of RP, and demonstrates how consanguinity could increase intrafamilial clustering of multiple hereditary diseases. Moreover, this report provides a unique opportunity to study the clinical implications of the coexistence of pathogenic mutations in two RP-causative genes in a human patient.
PMCID: PMC3718492  PMID: 23882135
7.  Ceramide Kinase-Like (CERKL) Interacts with Neuronal Calcium Sensor Proteins in the Retina in a Cation-Dependent Manner 
Purpose.
CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina.
Methods.
CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay.
Results.
We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cation-binding domains (EF-hands 2–4).
Conclusions.
This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca2+ signaling, providing important clues for future research required in this direction.
Ceramide kinase-like (CERKL) interacts with several neuronal calcium sensor proteins, including GCAP1, GCAP2, and recoverin. This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca2+ signaling.
doi:10.1167/iovs.12-9770
PMCID: PMC3394741  PMID: 22678504
8.  Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss 
PLoS ONE  2012;7(12):e51566.
We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.
doi:10.1371/journal.pone.0051566
PMCID: PMC3520954  PMID: 23251578
9.  Evidence for a founder mutation causing DFNA5 hearing loss in East Asians 
Journal of human genetics  2009;55(1):59-62.
Mutations in the DFNA5 gene are known to cause autosomal dominant non-syndromic hearing loss (ADNSHL). To date, five DFNA5 mutations have been reported, all of which were different in the genomic level. In this study, we ascertained a Korean family with autosomal dominant, progressive and sensorineural hearing loss and performed linkage analysis that revealed linkage to the DFNA5 locus on chromosome 7. Sequence analysis of DFNA5 identified a 3-bp deletion in intron 7 (c.991-15_991-13del) as the cause of hearing loss in this family. As the same mutation had been reported in a large Chinese family segregating DFNA5 hearing loss, we compared their DFNA5 mutation-linked haplotype with that of the Korean family. We found a conserved haplotype, suggesting that the 3-bp deletion is derived from a single origin in these families. Our observation raises the possibility that this mutation may be a common cause of autosomal dominant progressive hearing loss in East Asians.
doi:10.1038/jhg.2009.114
PMCID: PMC3433838  PMID: 19911014
DFNA5; founder effect; hearing loss; mutation; non-syndromic
10.  Novel mutations of MYO7A and USH1G in Israeli Arab families with Usher syndrome type 1 
Molecular Vision  2011;17:3548-3555.
Purpose
This study investigated the genetic basis for Usher syndrome type 1 (USH1) in four consanguineous Israeli Arab families.
Methods
Haplotype analysis for all known USH1 loci was performed in each family. In families for which haplotype analysis was inconclusive, we performed genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array. For mutation analysis, specific primers were used to PCR amplify the coding exons of the MYO7A, USH1C, and USH1G genes including intron-exon boundaries. Mutation screening was performed with direct sequencing.
Results
A combination of haplotype analysis and genome-wide homozygosity mapping indicated linkage to the USH1B locus in two families, USH1C in one family and USH1G in another family. Sequence analysis of the relevant genes (MYO7A, USH1C, and USH1G) led to the identification of pathogenic mutations in all families. Two of the identified mutations are novel (c.1135–1147dup in MYO7A and c.206–207insC in USH1G).
Conclusions
USH1 is a genetically heterogenous condition. Of the five USH1 genes identified to date, USH1C and USH1G are the rarest contributors to USH1 etiology worldwide. It is therefore interesting that two of the four Israeli Arab families reported here have mutations in these two genes. This finding further demonstrates the unique genetic structure of the Israeli population in general, and the Israeli Arab population in particular, which due to high rates of consanguinity segregates many rare autosomal recessive genetic conditions.
PMCID: PMC3250379  PMID: 22219650
11.  Double gene deletion reveals the lack of cooperation between claudin 11 and claudin 14 tight junction proteins 
Cell and tissue research  2008;333(3):427-438.
Summary
Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. Selectivity and specificity of TJ strands are determined by the type of claudins present. It is thus important to understand the cooperation between different claudins in various tissues. To study the possible cooperation between claudin 11 and claudin 14 we generated claudin11/claudin 14 double deficient mice. These mice exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits and male sterility. In the kidney we found that these two claudins have distinct and partially overlapping expression patterns. Claudin 11 is located in both the proximal and the distal convoluted tubules, while claudin 14 is located in both the thin descending and the thick ascending limbs of the loop of Henle, as well as in the proximal convoluted tubules. Although daily urinary excretion of Mg++, and to a lesser extent of Ca++, tended to be higher in claudin11/claudin 14 double mutants, these changes did not reach statistical significance comparing to wt animals. These findings suggest that under normal conditions co-deletion of claudin11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished will provide further insight into the complex interactions between claudin proteins in various physiological systems.
doi:10.1007/s00441-008-0621-9
PMCID: PMC2943968  PMID: 18663477
tight junction; claudin 11; claudin 14; knockout mouse
12.  Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations 
Journal of medicinal chemistry  2009;52(9):2836-2845.
Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.
doi:10.1021/jm801640k
PMCID: PMC2832307  PMID: 19309154
Aminoglycosides; Genetic Diseases; Nonsense Mutations; Translational Therapy; Cystic Fibrosis
13.  Spatiotemporal expression pattern of ceramide kinase-like in the mouse retina 
Molecular Vision  2010;16:2539-2549.
Purpose
The CERKL gene encodes for ceramide kinase-like, a novel protein of unknown function. CERKL mutations are associated with a severe retinal phenotype. The purpose of this work was to investigate alternative splicing, and the temporal and spatial expression pattern of CERKL in the mouse retina.
Methods
Reverse Transcription-Polymerase Chain Reaction (RT–PCR) analysis of mouse retina RNA was used to study the expression of Cerkl at various developmental time points, and to identify its various splice-isoforms. A specific anti-CERKL antibody was developed and used for immunostaining to study the localization of the endogenous CERKL protein in retina-derived cell lines and in the mouse retina.
Results
Cerkl is expressed in the mouse eye as early as embryonic day 14. A total of seven different Cerkl splice-isoforms were identified in the mouse retina. The subcellular localization of CERKL in retina-derived cell lines is variable: CERKL is diffusely distributed in the cytoplasm, and in many cells, it is highly concentrated in the perinuclear region. In most, but not all cells, CERKL is also highly concentrated in the nucleus. In the mouse retina, CERKL is located in the ganglion cell layer, in amacrine cells of the inner nuclear layer, and in photoreceptors. CERKL is highly expressed in cone photoreceptors; however, its expression level in rod photoreceptors is very low. In cultured cells, CERKL is detected in the nucleus, but in retinal cells in situ, it is mostly located in the cytoplasm.
Conclusions
The expression of Cerkl in both mature and embryonic mouse retina and the severe retinal phenotype associated with human CERKL mutations indicate that this gene plays a crucial role in retinal activity, and that it may be important for retinal development as well. The high expression level of CERKL in cones correlates with the CERKL-associated phenotype in humans. Whether nucleocytoplasmic transport of CERKL actually occurs in vivo under certain conditions and its functional significance remain to be discovered.
PMCID: PMC3000240  PMID: 21151604
15.  A novel splice-site mutation of TULP1 underlies severe early-onset retinitis pigmentosa in a consanguineous Israeli Muslim Arab family 
Molecular Vision  2008;14:675-682.
Purpose
To investigate the genetic basis for autosomal recessive severe early-onset retinitis pigmentosa (RP) in a consanguineous Israeli Muslim Arab family.
Methods
Haplotype analysis for all known genes underlying autosomal recessive RP was performed. Mutation screening of the underlying gene was done by direct sequencing. An in vitro splicing assay was used to evaluate the effect of the identified mutation on splicing.
Results
Haplotype analysis indicated linkage to the Tubby-like protein 1 (TULP)1 gene. Direct sequencing revealed a homozygous single base insertion, c.1495+2_1495+3insT, located in the conserved donor splice-site of intron 14. This mutation co-segregated with the disease, and was not detected in 114 unrelated Israeli Muslim Arab controls. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing.
Conclusions
To date, 22 distinct pathogenic mutations of TULP1 have been reported in patients with early-onset RP or Leber congenital amaurosis. Here we report a novel splice-site mutation of TULP1, c.1495+2_1495+3insT, underlying autosomal recessive early-onset RP in a consanguineous Israeli Muslim Arab family. This report expands the spectrum of pathogenic mutations of the TULP1 gene.
PMCID: PMC2329669  PMID: 18432314

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