Gene therapy of hematological disorders: current challenges.

Recent advances in genetic engineering technology and stem cell biology have spurred great interest in developing gene therapies for hereditary, as well as acquired hematological disorders. Currently, hematopoietic stem cell transplantation is used to cure disorders such as hemoglobinopathies and primary immunodeficiencies; however, this method is limited by the availability of immune-matched donors. Using autologous cells coupled with genome editing bypasses this limitation and therefore became the focus of many research groups aiming to develop efficient and safe genomic modification. Hence, gene therapy research has witnessed a noticeable growth in recent years with numerous successful achievements; however, several challenges have to be overcome before gene therapy becomes widely available for patients. In this review, I discuss tools used in gene therapy for hematological disorders, choices of target cells, and delivery vehicles with emphasis on current hurdles and attempts to solve them, and present examples of successful clinical trials to give a glimpse of current progress.

Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson's disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

Missense mutations in TCF8 cause late-onset Fuchs corneal dystrophy and interact with FCD4 on chromosome 9p.

Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay. Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an additional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic variants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expressivity of the phenotype.

Loss of ERLIN2 function leads to juvenile primary lateral sclerosis.

OBJECTIVE: Primary lateral sclerosis (PLS) is a motor neuron disorder that exclusively affects upper motor neurons leading to their degeneration. Mutations in the ALS2 gene encoding the protein Alsin have been described previously in the juvenile form of the disease. In this study, we identify mutation of the ERLIN2 gene in juvenile PLS patients and describe an in vitro model for loss of ERLIN2 function. METHODS: Single nucleotide polymorphism arrays were used for homozygosity mapping. DNA sequencing of candidate genes was used to detect the underlying mutation. Level of ERLIN2 mRNA was measured by quantitative real time polymerase chain reaction. Knocking down ERLIN2 in NSC34 cells was accomplished by short-hairpin RNA interference. RESULTS: We identified a splice junction mutation in the ERLIN2 gene-a component of the endoplasmic reticulum (ER) lipid rafts-that resulted in abnormal splicing of ERLIN2 transcript and nonsense-mediated decay of ERLIN2 mRNA. Knocking down ERLIN2 in NSC34 cells suppressed their growth in culture. INTERPRETATION: Recently, we found that mutation of SIGMAR1, a component of ER lipid rafts, leads to juvenile amyotrophic lateral sclerosis. The identification of mutation in another component of the ER lipid rafts in juvenile PLS patients emphasizes their role in motor neuron function. Furthermore, the discovered effect of ERLIN2 loss on cell growth may advance understanding of the mechanism behind motor neuron degeneration in PLS.

A mutation in sigma-1 receptor causes juvenile amyotrophic lateral sclerosis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the brain and spinal cord, leading to muscle weakness and eventually death from respiratory failure. ALS is familial in about 10% of cases, with SOD1 mutations accounting for 20% of familial cases. Here we describe a consanguineous family segregating juvenile ALS in an autosomal recessive pattern and describe the genetic variant responsible for the disorder. METHODS: We performed homozygosity mapping and direct sequencing to detect the genetic variant and tested the effect of this variant on a motor neuron-like cell line model (NSC34) expressing the wild-type or mutant gene. RESULTS: We identified a shared homozygosity region in affected individuals that spans ~120 kbp on chromosome 9p13.3 containing 9 RefSeq genes. Sequencing the SIGMAR1 gene revealed a mutation affecting a highly conserved amino acid located in the transmembrane domain of the encoded protein, sigma-1 receptor. The mutated protein showed an aberrant subcellular distribution in NSC34 cells. Furthermore, cells expressing the mutant protein were less resistant to apoptosis induced by endoplasmic reticulum stress. INTERPRETATION: Sigma-1 receptors are known to have neuroprotective properties, and recently Sigmar1 knockout mice have been described to have motor deficiency. Our findings emphasize the role of sigma-1 receptors in motor neuron function and disease.

Mutations in C2orf37, encoding a nucleolar protein, cause hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome.

Hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome (also referenced as Woodhouse-Sakati syndrome) is a rare autosomal recessive multisystemic disorder. We have identified a founder mutation consisting of a single base-pair deletion in C2orf37 in eight families of Saudi origin. Three other loss-of-function mutations were subsequently discovered in patients of different ethnicities. The gene encodes a nucleolar protein of unknown function, and the cellular phenotype observed in patient lymphoblasts implicates a role for the nucleolus in the pathogenesis of this disease. Our findings expand the list of human disorders linked to the nucleolus and further highlight the developmental and/or maintenance functions of this organelle.

Missense mutations in the sodium borate cotransporter SLC4A11 cause late-onset Fuchs corneal dystrophy.

Homozygous mutations in the Borate Cotransporter SLC4A11 cause two early-onset corneal dystrophies: congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome. More recently, four sporadic patients with late-onset Fuchs corneal dystrophy (FCD), a common age-related disorder, were also reported to harbor heterozygous mutations at this locus. We therefore tested the hypothesis that SLC4A11 contributes to FCD and asked whether mutations in SLC4A11 are responsible for familial cases of late-onset FCD. We sequenced SLC4A11 in 192 sporadic and small nuclear late-onset FCD families and found seven heterozygous missense novel variations that were absent from ethnically matched controls. Familial data available for one of these mutations showed segregation under a dominant model in a three-generational family. In silico analyses suggested that most of these substitutions are intolerant, whereas biochemical studies of the mutant protein indicated that these alleles impact the localization and/or posttranslational modification of the protein. These results suggest that heterozygous mutations in SLC4A11 are modest contributors to the pathogenesis of adult FCD, suggesting a causality continuum between FCD and CHED. Taken together with a recent model between FCD and yet another early onset corneal dystrophy, PPCD, our data suggest a shared pathomechanism and genetic overlap across several corneal dystrophies.

Autosomal recessive ADCY5-Related dystonia and myoclonus: Expanding the genetic spectrum of ADCY5-Related movement disorders.

INTRODUCTION: ADCY5-related hyperkinesia encompasses a heterogeneous group of phenotypes, including paroxysmal chorea, myoclonus, and dystonia. The disease is attributed to mutations of ADCY5, which encodes an adenylate cyclase enzyme. The disease can occur in a sporadic or familial pattern. With exception of one study, all reports on familial ADCY5-related hyperkinesia were associated with an autosomal dominant inheritance. Herein, we describe a native Arabian Bedouin family with an autosomal recessive ADCY5-related disorder and expand the genotypic and phenotypic spectrum of this disorder. METHODS: The pedigree included 4 generations of a family with 6 affected individuals. The patients were examined clinically and radiologically. Homozygosity mapping and Whole Exome Sequencing (WES) were used to identify a variant, predicted to be pathogenic, which segregated with disease in this family. RESULTS: All patients presented with early-onset dystonia and myoclonus. The patients had delayed motor and language milestones, axial hypotonia, severe anxiety, social phobia, and isolation. One patient had dilated cardiomyopathy. WES of one affected individual revealed a novel homozygous missense mutation (c.1762G > A, p.D588N) of ADCY5, that segregated with disease in an autosomal recessive manner, and was absent in more than 1000 ethnically-matched chromosomes. The mutation replaces a highly conserved nucleotide and is predicted to be deleterious. CONCLUSION: This study reports the second family with autosomal recessive childhood-onset ADCY5-related disorder and expands our understanding of phenotype/genotype correlations of this disorder.

A novel KERA mutation associated with autosomal recessive cornea plana.

PURPOSE: To report a novel KERA mutation associated with autosomal recessive cornea plana in members of a nuclear family and to describe their ophthalmic phenotypes. METHODS: Ophthalmic examination, biometry, and direct sequencing of KERA. RESULTS: Five of the 6 siblings were affected and had small flat corneas, variable anterior chamber depths, and short axial lengths. The remaining brother and the 2 parents had normal ophthalmic examinations. Genetic testing revealed a novel homozygous nonsense mutation in exon 3 [937C>T] in the clinically affected individuals. The clinically unaffected parents were confirmed as carriers. The clinically unaffected sibling had no KERA mutation. This mutation leads to replacement of an arginine by a stop codon at position 313 of keratocan protein. CONCLUSIONS: This novel point mutation in KERA is the fourth thus far described. The ocular phenotype is characteristic of autosomal recessive cornea plana.

A novel missense Norrie disease mutation associated with a severe ocular phenotype.

Clinical findings and pedigree analysis led to the diagnosis of severe Norrie disease in two brothers. DNA sequencing demonstrated a novel missense mutation (703G>T) that significantly alters predicted protein structure. Less severe retinal developmental disease may be associated with milder mutations in the Norrie disease gene.

Mutation of fibulin-1 causes a novel syndrome involving the central nervous system and connective tissues.

Fibulin-1 is an extracellular matrix protein that has an important role in the structure of elastic fibers and basement membranes of various tissues. Using homozygosity mapping and exome sequencing, we discovered a missense mutation, p.(Cys397Phe), in fibulin-1 in three patients from a consanguineous family presented with a novel syndrome of syndactyly, undescended testes, delayed motor milestones, mental retardation and signs of brain atrophy. The mutation discovered segregated with the phenotype and was not found in 374 population-matched alleles. The affected cysteine is highly conserved across vertebrates and its mutation is predicted to abolish a disulfide bond that defines the tertiary structure of fibulin-1. Our findings emphasize the crucial role fibulin-1 has in development of the central nervous system and various connective tissues.

Propionic acidemia associated with visual hallucinations.

Propionic acidemia, an autosomal recessive disorder, is a common form of organic aciduria resulting from the deficiency of propionyl-CoA carboxylase. It is characterized by frequent and potentially lethal episodes of metabolic acidosis often accompanied by hyperammonemia. A wide range of brain abnormalities have been reported in propionic acidemia. We report recurrent visual hallucinations in 2 children with propionic acidemia. Four visual hallucination events were observed in the 2 patients. Three episodes were preceded by an intercurrent illness, and 2 were associated with mild metabolic decompensation. The 2 events in one patient were associated with a seizure disorder with abnormal electroencephalogram. Brain magnetic resonance imaging showed abnormal basal ganglia and faint temporo-occipital swelling bilaterally. This is probably the first report of visual hallucinations in propionic acidemia and should alert the treating clinicians to look for visual hallucinations in patients with organic acidurias, especially in an unusually anxious child.

Age-severity relationships in families linked to FCD2 with retroillumination photography.

PURPOSE: Fuchs corneal dystrophy (FCD) is a progressive disorder of the corneal endothelium and is pathologically defined by the presence of guttae, which are excrescences of the Descemet membrane. The present study was undertaken to investigate the age-severity relationship of the FCD2-linked disease phenotype using retroillumination photography and to compare it with the characteristics of FCD1. METHODS: Two large families with multiple affected members were recruited. Exclusion analyses of the known late-onset FCD loci were completed with closely spaced STR markers, whereas genes associated with early- and late-onset FCD were investigated by bidirectional sequencing. Haplotypes were constructed, and two-point LOD scores were calculated. To document age-severity relationships, retroillumination photographs were acquired from members of both families. RESULTS: Parametric linkage and haplotype analysis mapped both families to FCD2 with significant two-point LOD scores. A total of 70,249 guttae were counted in 14 persons from both families. A significant increase in guttae density in the inferotemporal region (P = 0.016) was observed, a pattern similarly observed in a family linked to FCD1. Similarly, FCD2-linked families display an exponential trend in severity with age, as was observed in a family linked to FCD1. Finally, comparison of FCD1 and FCD2 exponential models suggested that the FCD1 phenotype is significantly more severe (P = 0.01). CONCLUSIONS: A combination of genetic mapping and retroillumination photography was used to quantify the severity of the disease phenotype associated with FCD2 and to compare it to the disease characteristics of FCD1. These data suggest that this approach might have sufficient resolution to discriminate between discrete genetic FCD backgrounds, which will potentially aid in patient management.

Linkage of a mild late-onset phenotype of Fuchs corneal dystrophy to a novel locus at 5q33.1-q35.2.

PURPOSE: To identify the disease locus associated with autosomal dominant Fuchs corneal dystrophy (FCD) in a large family and to compare the progression of severity in families mapped to the FCD1 and FCD2 loci. METHODS: Seventeen individuals in a large family were examined by slit lamp biomicroscopy. Blood samples were collected, DNA was extracted, and a genome-wide scan was performed with a microarray SNP chip. After initial generation of a genome-wide, two-point LOD score, linkage was confirmed and the critical interval was established by genotyping of short tandem repeat (STR) microsatellite markers. RESULTS: A genome-wide linkage scan localized the disease interval to the long arm of chromosome 5, with a maximum two-point parametric LOD score of 3.41. Haplotype analyses refined the critical interval to 5q33.1-q35.2, spanning a 27-Mb (29-cM) region. Clinical examination of affected individuals in this family revealed an early onset of FCD at approximately age 40, after which progression of the disease was significantly attenuated compared to the FCD1- and FCD2-linked families. CONCLUSIONS: Late-onset FCD is linked to a novel locus on 5q33.1-q35.2 and is associated with a milder severity in age at onset and rate of progression than the FCD1 and FCD2 loci. Correlation of individual genotypes with unique rates of disease progression will provide important tools for disease management, as well as for identifying the underlying genetic lesion, offer insight into the pathomechanism of FCD.

Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b.

Despite its recognized utility, the extent to which evolutionary sequence conservation-based approaches may systematically overlook functional noncoding sequences remains unclear. We have tiled across sequence encompassing the zebrafish phox2b gene, ultimately evaluating 48 amplicons corresponding to all noncoding sequences therein for enhancer activity in zebrafish. Post hoc analyses of this interval utilizing five commonly used measures of evolutionary constraint (AVID, MLAGAN, SLAGAN, phastCons, WebMCS) demonstrate that each systematically overlooks regulatory sequences. These established algorithms detected only 29%-61% of our identified regulatory elements, consistent with the suggestion that many regulatory sequences may not be readily detected by metrics of sequence constraint. However, we were able to discriminate functional from nonfunctional sequences based upon GC composition and identified position weight matrices (PWM), demonstrating that, in at least one case, deleting sequences containing a subset of these PWMs from one identified regulatory element abrogated its regulatory function. Collectively, these data demonstrate that the noncoding functional component of vertebrate genomes may far exceed estimates predicated on evolutionary constraint.