Proof of concept for MERTK gene replacement therapy has been demonstrated utilizing different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa (RP) that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP compliant safety evaluations of an AAV2 vector expressing human MERTK cDNA driven by the RPE-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram responses in treated eyes compared to contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to Balanced Salt Solution (BSS) control injected eyes indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector injected eyes were normal compared to controls. Evaluation of blood smears showed the lack of transformed cells in the. All injected eyes and day 1 blood samples were positive for vector genomes and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.
Clinical immunology has traditionally relied on accurate phenotyping of the patient’s immune dysfunction for the identification of a candidate gene or genes for sequencing and molecular confirmation. Although this is also true for other branches of medicine, the marked variability in immune-related phenotypes and the highly complex network of molecules that confer normal host immunity are challenges that clinical immunologists often face in their quest to establish a specific genetic diagnosis.
We sought to identify the underlying genetic cause in a consanguineous family with chronic inflammatory bowel disease–like disorder and combined immunodeficiency.
We performed exome sequencing followed by autozygome filtration.
A truncating mutation in LPS-responsive beige-like anchor (LRBA), which abolished protein expression, was identified as the most likely candidate variant in this family.
The combined exome sequencing and autozygosity mapping approach is a powerful tool in the study of atypical immune dysfunctions. We identify LRBA as a novel immunodeficiency candidate gene the precise role of which in the immune system requires future studies.
LPS-responsive beige-like anchor (LRBA); chronic diarrhea; common variable immunodeficiency; autoimmunity
Bardet–Biedl syndrome (BBS) is a model disease for ciliopathy in humans. The remarkable genetic heterogeneity that characterizes this disease is consistent with accumulating data on the interaction between the proteins encoded by the 14 BBS genes identified to date. Previous reports suggested that such interaction may also extend to instances of oligogenic inheritance in the form of triallelism which defies the long held view of BBS as an autosomal recessive disease. In order to investigate the magnitude of triallelism in BBS, we conducted a comprehensive analysis of all 14 BBS genes as well as the CCDC28B-modifier gene in a cohort of 29 BBS families, most of which are multiplex. Two in trans mutations in a BBS gene were identified in each of these families for a total of 20 mutations including 12 that are novel. In no instance did we observe two mutations in unaffected members of a given family, or observe the presence of a third allele that convincingly acted as a modifier of penetrance and supported the triallelic model of BBS. In addition to presenting a comprehensive genotype/phenotype overview of a large set of BBS mutations, including the occurrence of nonsyndromic retinitis pigmentosa in a family with a novel BBS9 mutation, our study argues in favor of straightforward autosomal recessive BBS in most cases.
epistasis; oligogenic; penetrance; modifiers
Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1−/− mice. Male Tysnd1−/− mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1−/− mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.
Peroxisomes are subcellular organelles that are present in almost all eukaryotic cells. The syllables “per-oxi” reflect the oxidative functions of these single-membrane-bound organelles in various metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids. In an earlier study we identified a protease named Tysnd1 that is specifically located in the peroxisomes and processes the enzymes catalyzing the peroxisomal β-oxidation of very-long-chain fatty acids. In this study, we identified two novel Tysnd1 substrates, Agps and Phyh, which are involved in plasmalogen synthesis and phytanic acid metabolism, respectively. To further investigate the in vivo function of Tysnd1, we analyzed Tysnd1 knock-out mice. Mice that lack Tysnd1 showed reduced peroxisomal β-oxidation activity and an altered plasmalogen composition, as well as an abnormal phytanic acid metabolism. Male infertility is one of the major phenotypic manifestations of Tysnd1 deficiency. Our data support the idea that Tysnd1 affects the localization and activity of some of its substrates inside peroxisomes. Altogether, our Tysnd1-deficient mouse model expands the current peroxisome biology knowledge with regard to the molecular pathogenic mechanisms that may be relevant to some patients with Zellweger syndrome spectrum disorders.
Hyperekplexia (HPX) is a rare non-epileptic disorder manifesting immediately after birth with exaggerated persistent startle reaction to unexpected auditory, somatosensory and visual stimuli, and non-habituating generalized flexor spasm in response to tapping of the nasal bridge (glabellar tap) which forms its clinical hallmark. The course of the disease is usually benign with spontaneous amelioration with age. The disorder results from aberrant glycinergic neurotransmission, and several mutations were reported in the genes encoding glycine receptor (GlyR) α1 and β subunits, glycine transporter GlyT2 as well as two other proteins involved in glycinergic neurotransmission gephyrin and collybistin.
The phenotype of six newborns, belonging to Saudi Arabian kindred with close consanguineous marriages, who presented with hyperekplexia associated with severe brain malformation, is described. DNA samples were available from two patients, and homozygosity scan to determine overlap with known hyperkplexia genes was performed.
The kindred consisted of two brothers married to their cousin sisters, each with three affected children who presented antenatally with excessive fetal movements. Postnatally, they were found to have microcephaly, severe hyperekplexia and gross brain malformation characterized by severe simplified gyral pattern and cerebellar underdevelopment. The EEG was normal and they responded to clonazepam. All of the six patients died within six weeks. Laboratory investigations, including metabolic screen, were unremarkable. None of the known hyperkplexia genes were present within the overlapping regions of homozygosity between the two patients for whom DNA samples were available.
We present these cases as a novel syndrome of lethal familial autosomal recessive hyperekplexia associated with microcephaly and severe brain malformation.
Hyperekplexia; Microcephaly; Simplified gyral pattern; Cerebellar underdevelopment; Autosomal recessive
The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis.
3MC syndrome has been proposed as a unifying term to integrate the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders of unknown cause comprise a spectrum of developmental features including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability, and genital, limb and vesicorenal anomalies. In a cohort of eleven 3MC families, we identified two mutated genes COLEC11 and MASP1 both of which encode proteins within the lectin complement pathway (CL-K1 and MASP-1 & −3 respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney, and vertebral bodies. Zebrafish morphants develop pigment defects and severe craniofacial abnormalities.
Here, we show that CL-K1 serves as a key guidance cue for neural crest cell migration thus demonstrating for the first time, a role for complement pathway factors in fundamental developmental processes and the origin of 3MC syndrome.
Hereditary Spastic Paraplegia (HSP) is a clinically and genetically heterogeneous group of neurological disorders that are characterized by progressive spasticity of the lower extremities. We describe an extended consanguineous Saudi family in which HSP is linked to SPG18, a previously reported autosomal recessive locus, and show that it is associated with a nullimorphic deletion of ERLIN2, a component of endoplasmic reticulum associated degradation. This finding adds to the growing diversity of cellular functions that are now known to be involved in the maintenance of the corticospinal tract neurons.
ERAD; Aphasia; Intellectual disability; ERLIN2
Bardet–Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.
To determine the genetic and genomic alterations underlying classic aniridia in Saudi Arabia, a region with social preference for consanguineous marriage.
Prospective study of consecutive patients referred to a pediatric ophthalmologist in Saudi Arabia (2005–2009). All patients had paired box gene 6 (PAX6) analysis (sequencing and multiplex ligation-dependent probe amplification analysis if sequencing was normal). If PAX6 analysis was negative, the following were performed: candidate gene sequencing (forkhead box C1 [FOXC1], paired-like homeodomain transcription factor 2 [PITX2], cytochrome P450, family 1, subfamily B [CYP1B1], paired-like homeodomain transcription factor 3 [PITX3], and v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog [MAF]) and molecular karyotyping by array competitive genomic hybridization (250K single nucleotide polymorphism (SNP) arrays).
All 12 probands (4 months – 25 years of age; four boys and eight girls) had lens opacity and foveal hypoplasia in addition to no grossly visible iris. Four cases were familial. All cases were products of consanguineous unions except for three, one of which was endogamous. Heterozygous PAX6 mutations (including two novel mutations) were detectable in all but two cases, both of which were sporadic. In one of these two cases, the phenotype segregated with homozygosity for a previously-reported pathogenic missense FOXC1 variant (p.P297S) when homozygosity for chromosome 11q24.2 deletion (chr11:125,001,547–125,215,177 [rs114259885; rs112291840]) was also present. In the other, no genetic or genomic abnormalities were found.
The classic aniridia phenotype in Saudi Arabia is typically caused by heterozygous PAX6 mutations, even in the setting of enhanced homozygosity from recent shared parental ancestry. For PAX6-negative cases, interaction between missense variation in an anterior segment developmental gene and copy number variation elsewhere in the genome may be a potential mechanism for the phenotype.
To clinically and genetically characterize a distinct phenotype of congenital megalocornea (horizontal corneal diameter ≥13 mm) with secondary glaucoma from spherophakia and/or ectopia lentis during childhood in affected Saudi families.
Clinical exam, homozygosity scan, and candidate gene analysis.
From 2005 to 2010, eight affected individuals from three consanguineous families were identified. In addition to congenital megalocornea, affected children presented with secondary glaucoma from spherophakia and/or ectopia lentis. One member from each family developed spontaneous complete crystalline lens dislocation into the anterior chamber with associated acute glaucoma during early childhood. Older individuals had phenotypes that would have suggested prior uncontrolled primary congenital/infantile glaucoma had past ophthalmic and/or family histories not been available. Homozygosity mapping performed for the first two families suggested the candidate gene latent transforming growth factor-beta-binding protein 2 (LTBP2), which when sequenced revealed a novel homozgyous mutation that segregated with the phenotype in each family (p.S338PfsX4 [c.1012delT], p.Q1619X[(c.4855C>T]). LTBP2 sequencing in the third family revealed a third novel homozygous mutation (p.C1438Y [c.4313G>A]).
Congenital megalocornea with childhood secondary glaucoma from spherophakia and/or ectopia lentis is a distinct condition caused by recessive LTBP2 mutations that needs to be distinguished from buphthalmos secondary to primary congenital/infantile glaucoma because typical initial surgical treatment is lens removal in the former and angle surgery in the latter. Complete dislocation of the crystalline lens into the anterior chamber during early childhood can occur in young children with this unique phenotype.
In an effort to discover genes important for human development, we have ascertained patients with congenital anomalies and cytogenetically balanced chromosomal rearrangements. Herein, we report a four year-old girl with profound deafness, a history of feeding difficulties, dysmorphism, strabismus, developmental delay, and an apparently balanced de novo paracentric chromosome 5 inversion, inv(5)(q15q33.2). Molecular cytogenetic analysis of the inversion revealed the presence of microdeletions of approximately 400-500 kb at or near both breakpoints. The 5q15 microdeletion completely removes the nuclear receptor NR2F1 (COUP-TFI) from the inverted chromosome 5. We propose haploinsufficiency of NR2F1 to be the cause of the patient's deafness and many of the other associated anomalies based on striking similarity with the Nr2f1 null mouse. Additionally, this study further highlights the need for high resolution analysis of clinical samples with chromosomal rearrangements as associated deletions may be primarily responsible for the clinical features of these patients.
NR2F1; deafness; chromosomal inversion; microdeletion; FISH; 5q
To describe the finding of a novel calcium binding protein 4 (CABP4) mutation in a family with Leber congenital amaurosis (LCA) phenotype.
Homozygosity mapping was performed in a consanguineous family with four affected members originally referred as cases of LCA. Detailed electroretinographic recordings were obtained.
A novel homozygous single base-pair insertion was identified in all four siblings. The patients had an LCA-like phenotype, including either flat or greatly diminished electroretinographic activity.
This report significantly expands on the phenotype associated with calcium binding protein 4 mutations, which has so far been limited to congenital stationary night blindness, and further demonstrates how molecular data often blur the boundaries between what are believed to be clinically distinct retinal disorders.
To report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene.
A consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations.
Analysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod–cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina.
Mutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.
To catalog mutations that underlie retinitis pigmentosa (RP) in Saudi Arabia using a representative sample.
Fifty-two patients with RP were recruited and their homozygosity mapping, with or without linkage analysis, was used to suggest the causative genes followed by bidirectional sequencing.
Mutations were identified in 94% of our study cohort, including seven that were novel.
Homozygosity mapping is an extremely robust approach in the study of retinitis pigmentosa in the setting of high rates of consanguinity. BBS3 mutations can rarely present as nonsyndromic RP.
We identified a homozygous missense mutation (c.196G → T) in fibroblast growth factor 3 (FGF3) in 21 affected individuals from a large extended consanguineous Saudi family, phenotypically characterized by autosomal recessive syndromic congenital sensorineural deafness, microtia and microdontia. All affected family members are descendents of a common ancestor who had lived six generations ago in a geographically isolated small village. This is the second report of FGF3 involvement in syndromic deafness in humans, and independently confirms the gene's positive role in inner ear development. The c.196G → T mutation results in substitution of glycine by cysteine at amino acid 66 (p.G66C). This residue is conserved in several species and across 18 FGF family members. Conserved glycine/proline residues are central to the ‘β-trefoil fold' characteristic of the secondary structure of FGF family proteins and substitution of these residues is likely to disrupt structure and consequently function.
syndromic deafness; sensorineural; Arab; consanguineous; FGF; congenital
To assess for γD-crystallin (CRYGD) mutation in 2 Saudi patients with cerulean cataract and in a brother of one of the patients who had coralliform cataract.
Patients and all of their available relatives underwent ophthalmic examination and subsequent sequencing of the candidate gene CRYGD.
In the first family, a 4-year-old boy with bilateral cerulean cataract and his 6-year-old brother with similar bluish lens discoloration but in a coralliform pattern were heterozygous for the p.P23T CRYGD mutation. Their father and 2 older brothers, all of whom underwent childhood cataract surgery, also harbored the mutation while the 2 asymptomatic immediate family members did not. In the second family, a 7-year-old girl with bilateral cerulean cataract was heterozygous for the same CRYGD mutation. Details of her family history were limited. The patients in the two families shared a common disease haplotype.
This first report of p.P23T CRYGD mutation underlying cerulean cataract in the Saudi population strongly supports the mutation’s relation with the phenotype. Coralliform cataract can represent variable expressivity for the same mutation rather than a distinct entity.
Complex central nervous system (CNS) malformations frequently coexist with other developmental abnormalities, but whether the associated defects share a common genetic basis is often unclear. We describe five individuals who share phenotypically related CNS malformations and in some cases urinary tract defects, and also haploinsufficiency for the NFIA transcription factor gene due to chromosomal translocation or deletion. Two individuals have balanced translocations that disrupt NFIA. A third individual and two half-siblings in an unrelated family have interstitial microdeletions that include NFIA. All five individuals exhibit similar CNS malformations consisting of a thin, hypoplastic, or absent corpus callosum, and hydrocephalus or ventriculomegaly. The majority of these individuals also exhibit Chiari type I malformation, tethered spinal cord, and urinary tract defects that include vesicoureteral reflux. Other genes are also broken or deleted in all five individuals, and may contribute to the phenotype. However, the only common genetic defect is NFIA haploinsufficiency. In addition, previous analyses of Nfia−/− knockout mice indicate that Nfia deficiency also results in hydrocephalus and agenesis of the corpus callosum. Further investigation of the mouse Nfia+/− and Nfia−/− phenotypes now reveals that, at reduced penetrance, Nfia is also required in a dosage-sensitive manner for ureteral and renal development. Nfia is expressed in the developing ureter and metanephric mesenchyme, and Nfia+/− and Nfia−/− mice exhibit abnormalities of the ureteropelvic and ureterovesical junctions, as well as bifid and megaureter. Collectively, the mouse Nfia mutant phenotype and the common features among these five human cases indicate that NFIA haploinsufficiency contributes to a novel human CNS malformation syndrome that can also include ureteral and renal defects.
Central nervous system (CNS) and urinary tract abnormalities are common human malformations, but their variability and genetic complexity make it difficult to identify the responsible genes. Analysis of human chromosomal abnormalities associated with such disorders offers one approach to this problem. In five individuals described herein, a novel human syndrome that involves both CNS and urinary tract defects is associated with chromosomal disruption or deletion of NFIA, encoding a member of the Nuclear Factor I (NFI) family of transcription factors. This syndrome includes brain abnormalities (abnormal corpus callosum, hydrocephalus, ventriculomegaly, and Chiari type I malformation), spinal abnormalities (tethered spinal cord), and urinary tract abnormalities (vesicoureteral reflux). Nfia disruption in mice was already known to cause hydrocephalus and abnormal corpus callosum, and is now shown to exhibit renal defects and disturbed ureteral development. Other genes besides NFIA are also disrupted or deleted and may contribute to the observed phenotype. However, loss of one copy of NFIA is the only genetic defect common to all five patients. The authors thus provide evidence that genetic loss of NFIA contributes to a distinct CNS malformation syndrome with urinary tract defects of variable penetrance.
Meckel–Gruber syndrome (MKS) is a genetically heterogeneous severe ciliopathy characterised by early lethality, occipital encephalocele, polydactyly, and polycystic kidney disease.
To report genetic analysis results in two families in which all known MKS diseases genes have been excluded.
In two consanguineous families with classical MKS in which autozygome-guided sequencing of previously reported MKS genes was negative, we performed exome sequencing followed by autozygome filtration.
We identified one novel splicing mutation in TMEM231, which led to complete degradation of the mutant transcript in one family, and a novel missense mutation in the other, both in the homozygous state.
TMEM231 represents a novel MKS locus. The very recent identification of TMEM231 mutations in Joubert syndrome supports the growing appreciation of the overlap in the molecular pathogenesis between these two ciliopathies.
TMEM231; Joubert; Ciliopathy; Autozygome; Exome