Sequencing of the IL6 gene in a case-control study of cerebral palsy in children.

BACKGROUND: Cerebral palsy (CP) is a group of nonprogressive disorders of movement and posture caused by abnormal development of, or damage to, motor control centers of the brain. A single nucleotide polymorphism (SNP), rs1800795, in the promoter region of the interleukin-6 (IL6) gene has been implicated in the pathogenesis of CP by mediating IL-6 protein levels in amniotic fluid and cord plasma and within brain lesions. This SNP has been associated with other neurological, vascular, and malignant processes as well, often as part of a haplotype block. METHODS: To refine the regional genetic association with CP, we sequenced (Sanger) the IL6 gene and part of the promoter region in 250 infants with CP and 305 controls. RESULTS: We identified a haplotype of 7 SNPs that includes rs1800795. In a recessive model of inheritance, the variant haplotype conferred greater risk (OR = 4.3, CI = [2.0-10.1], p = 0.00007) than did the lone variant at rs1800795 (OR = 2.5, CI = [1.4-4.6], p = 0.002). The risk haplotype contains one SNP (rs2069845, CI = [1.2-4.3], OR = 2.3, p = 0.009) that disrupts a methylation site. CONCLUSIONS: The risk haplotype identified in this study overlaps with previously identified haplotypes that include additional promoter SNPs. A risk haplotype at the IL6 gene likely confers risk to CP, and perhaps other diseases, via a multi-factorial mechanism.

Aggregation of multiple sclerosis genetic risk variants in multiple and single case families.

OBJECTIVE: Multiple sclerosis (MS) is a multifactorial neurologic disease characterized by modest but tractable heritability. Genome-wide association studies have identified and/or validated multiple polymorphisms in approximately 16 genes associated with susceptibility. We aimed at investigating the aggregation of genetic MS risk markers in individuals by comparing multiple- and single-case families. METHODS: A weighted log-additive integrative approach termed MS genetic burden (MSGB) was used to account for the well-established genetic variants from previous association studies and meta-analyses. The corresponding genetic burden and its transmission was analyzed in 1,213 independent MS families (810 sporadic and 403 multicase families). RESULTS: MSGB analysis demonstrated a higher aggregation of susceptibility variants in multicase compared to sporadic MS families. In addition, the aggregation of non-major histocompatibility complex single nucleotide polymorphisms depended neither on gender nor on the presence or absence of HLA-DRB1*15:01 alleles. Interestingly, although a greater MSGB in siblings of MS patients was associated with an increased risk of MS (odds ratio, 2.1; p = 0.001), receiver operating characteristic curves of MSGB differences between probands and sibs (area under the receiver operator curves, 0.57 [95% confidence interval, 0.53-0.61]) show that case-control status prediction of MS cannot be achieved with the currently available genetic data. INTERPRETATION: The primary interest in the MSGB concept resides in its capacity to integrate cumulative genetic contributions to MS risk. This analysis underlines the high variability of family load with known common variants. This novel approach can be extended to other genetically complex diseases. Despite the emphasis on assembling large case-control datasets, multigenerational, multiaffected families remain an invaluable resource for advancing the understanding of the genetic architecture of complex traits.

SMA Identified: Clinical and Molecular Findings From a Sponsored Testing Program for Spinal Muscular Atrophy in More Than 2,000 Individuals.

Background: Spinal muscular atrophy (SMA) linked to chromosome 5q is an inherited progressive neuromuscular disorder with a narrow therapeutic window for optimal treatment. Although genetic testing provides a definitive molecular diagnosis that can facilitate access to effective treatments, limited awareness and other barriers may prohibit widespread testing. In this study, the clinical and molecular findings of SMA Identified-a no-charge sponsored next-generation sequencing (NGS)-based genetic testing program for SMA diagnosis-are reported. Methods: Between March 2018 and March 2020, unrelated individuals who had a confirmed or suspected SMA diagnosis or had a family history of SMA were eligible. All individuals underwent diagnostic genetic testing for SMA at clinician discretion. In total, 2,459 individuals were tested and included in this analysis. An NGS-based approach interrogated sequence and copy number of SMN1 and SMN2. Variants were confirmed by multiplex ligation-dependent probe amplification sequencing. Individuals were categorized according to genetic test results: diagnostic (two pathogenic SMN1 variants), nearly diagnostic (SMN1 exon-7 deletion with a variant of uncertain significance [VUS] in SMN1 or SMN2), indeterminate VUS (one VUS in SMN1 or SMN2), carrier (heterozygous SMN1 deletion only), or negative (no pathogenic variants or VUS in SMN1 or SMN2). Diagnostic yield was calculated. Genetic test results were analyzed based on clinician-reported clinical features and genetic modifiers (SMN2 copy number and SMN2 c.859G>C). Results: In total, 2,459 unrelated individuals (mean age 24.3 ± 23.0 years) underwent diagnostic testing. The diagnostic yield for diagnostic plus nearly diagnostic results was 31.3% (n = 771/2,459). Age of onset and clinical presentation varied considerably for individuals and was dependent on SMN2 copy number. Homozygous deletions represented the most common genetic etiology (96.2%), with sequence variants also observed in probands with clinical diagnoses of SMA. Conclusions: Using a high-yield panel test in a no-charge sponsored program early in the diagnostic odyssey may open the door for medical interventions in a substantial number of individuals with SMA. These findings have potential implications for clinical management of probands and their families.

Genetic modification of the schisis phenotype in a mouse model of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an inherited form of macular degeneration that is caused by mutations in the retinoschisin (RS1) gene. In addition to macular degeneration, other major characteristics of XLRS include splitting of the retina (schisis) and impaired synaptic transmission as indicated by a reduction in the electroretinogram b-wave. It has been known that patients carrying RS1 mutations show a broad range of phenotypic variability. Interestingly, phenotypic variation is observed even among family members with the same RS1 mutation, suggesting the existence of genetic or environmental factors that contribute to the severity of XLRS. However, in the human population, the cause of phenotypic variability and the contribution of genetic modifiers for this relatively rare disease are difficult to study and poorly understood. In this study, using a mouse model for XLRS, we show that genetic factors can contribute to the severity of the retinoschisis phenotype. We report evidence of a major genetic modifier of Rs1, which affects the disease severity in these animals. A quantitative trait locus (QTL), named modifier of Rs1 1 (Mor1), is mapped on chromosome (Chr) 7. When homozygous, the Mor1 allele from the inbred mouse strain AKR/J diminishes the severity of the schisis phenotype in Rs1(tmgc1)/Y male and Rs1(tmgc1)/Rs1(tmgc1) female mice. We also show that the penetrance of the disease phenotype is affected by additional genetic factor(s). Our study suggests that multiple genetic modifiers could potentially be responsible for the phenotypic variation in human XLRS.

Diagnosing lysosomal storage disorders: mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry.

A Mutation in Syne2 Causes Early Retinal Defects in Photoreceptors, Secondary Neurons, and Müller Glia.

PURPOSE: The purpose of this study was to identify the molecular basis and characterize the pathological consequences of a spontaneous mutation named cone photoreceptor function loss 8 (cpfl8) in a mouse model with a significantly reduced cone electroretinography (ERG) response. METHODS: The chromosomal position for the recessive cpfl8 mutation was determined by DNA pooling and by subsequent genotyping with simple sequence length polymorphic markers in an F2 intercross phenotyped by ERG. Genes within the candidate region of both mutants and controls were directly sequenced and compared. The effects of the mutation were examined in longitudinal studies by light microscopy, marker analysis, transmission electron microscopy, and ERG. RESULTS: The cpfl8 mutation was mapped to Chromosome 12, and a premature stop codon was identified in the spectrin repeat containing nuclear envelope 2 (Syne2) gene. The reduced cone ERG response was due to a significant reduction in cone photoreceptors. Longitudinal studies of the early postnatal retina indicated that the cone photoreceptors fail to develop properly, rod photoreceptors mislocalize to the inner nuclear layer, and both rods and cones undergo apoptosis prematurely. Moreover, we observed migration defects of secondary neurons and ectopic Müller cell bodies in the outer nuclear layer in early postnatal development. CONCLUSIONS: SYNE2 is important for normal retinal development. We have determined that not only is photoreceptor nuclear migration affected, but also the positions of Müller glia and secondary neurons are disturbed early in retinal development. The cpfl8 mouse model will serve as an important resource for further examining the role of nuclear scaffolding and migration in the developing retina.

Diagnosis of lysosomal storage disorders: Gaucher disease.

Gaucher Disease (GD) is a progressive lysosomal storage disorder caused by deficiency of glucocerebrosidase (GBA). The clinical phenotype follows a spectrum ranging from severe early-onset to milder late-onset disease. The absence of neurological involvement defines GD type I, whereas neuronopathic features define GD type II and III. Early diagnosis may be important for timely initiation of enzyme replacement therapy to prevent disease complications, although the enzyme does not cross the blood brain barrier. Diagnosis of GD can be readily achieved by analysis of GBA in leukocytes, fibroblasts, and/or dried blood spots using fluorometric, microfluidic or mass spectrometry-based assays. Low GBA activities are typically confirmed through molecular analysis of the GBA gene. GBA analysis in dried blood spots may be attractive for high-throughput screening of at-risk individuals and/or newborn infants. The method detailed in this unit is based on GBA analysis by tandem mass spectrometry following incubation of dried blood spots with the GBA-specific substrate D-glucosyl-ß1-1'-N-dodecanoyl-D-erythro-sphingosine [C12-glucocerebroside (C36H69NO8)] and internal standard N-myristoyl-D-erythro-sphingosine [C14-ceramide (C32H63NO3)]. GBA activities in more than 2,000 newborn infants showed a mean of 22.0 ± 13.8 µmol/hr/liter (median: 19.9 µmol/hr/liter; 95% CI: 21.41-22.59 µmol/hr/liter). GBA activities in an adult population (n >1,200) showed generally lower enzyme activities than newborns, with a mean of 9.87 ± 9.35 µmol/hr/liter (median: 8.06 µmol/hr/liter). GBA activities in ten adult patients with confirmed GD were less than 4.2 µmol/hr/liter and in seven infants and children with GD less than 1.24 µmol/hr/liter. This method is robust, sensitive, and suitable for high-throughput analysis of hundreds of samples.

Diagnosing lysosomal storage disorders: mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay.

Tyrosinase is the modifier of retinoschisis in mice.

X-linked retinoschisis (XLRS) is a form of macular degeneration with a juvenile onset. This disease is caused by mutations in the retinoschisin (RS1) gene. The major clinical pathologies of this disease include splitting of the retina (schisis) and a loss in synaptic transmission. Human XLRS patients display a broad range in phenotypic severity, even among family members with the same mutation. This variation suggests the existence of genetic modifiers that may contribute to disease severity. Previously, we reported the identification of a modifier locus, named Mor1, which affects severity of schisis in a mouse model of XLRS (the Rs1tmgc1 mouse). Homozygosity for the protective AKR allele of Mor1 restores cell adhesion in Rs1tmgc1 mice. Here, we report our study to identify the Mor1 gene. Through collecting recombinant mice followed by progeny testing, we have localized Mor1 to a 4.4-Mb region on chromosome 7. In this genetic region, the AKR strain is known to carry a mutation in the tyrosinase (Tyr) gene. We observed that the schisis phenotype caused by the Rs1 mutation is rescued by a Tyr mutation in the C57BL/6J genetic background, strongly suggesting that Tyr is the Mor1 gene.

Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum.

Carbonic anhydrase related protein 8 (Car8) is known to be abundantly expressed in Purkinje cells (PCs), and its genetic mutation causes a motor coordination defect. To determine the underlying mechanism, we analyzed the mouse cerebellum carrying a Car8 mutation. Electrophysiological analysis showed that spontaneous excitatory transmission was largely diminished while paired pulse ratio at parallel fiber-PC synapses was comparable to wild-type, suggesting functional synapses have normal release probability but the number of functional synapses may be lower in mutants. Light microscopic study revealed an abnormal extension of climbing fibers to the distal PC dendrites. At the ultrastructural level, we found numerous PC spines not forming synapses primarily in distal dendrites and occasionally multiple spines contacting a single varicosity. These abnormalities of parallel fiber-PC synapses may underlie the functional defect in excitatory transmission. Thus, Car8 plays a critical role in synaptogenesis and/or maintenance of proper synaptic morphology and function in the cerebellum.

Reduced synaptic vesicle density and aberrant synaptic localization caused by a splice site mutation in the Rs1h gene.

X-linked retinoschisis (XLRS) is a common form of inherited macular degeneration caused by mutations in the RS1 gene. Whereas the role of RS1 has been implicated in the synaptic structure as well as layer organization in the retina, the pathological effect of a defective RS1 gene on the synaptic interaction between photoreceptor cells and second-order neurons has not been thoroughly investigated. In this study, we perform a detailed characterization of the retinal synaptic phenotypes caused by a splice site mutation in the murine RS1 homolog (Rs1h(tmgc1)). Electron microscopic analysis showed that presynaptic terminals of photoreceptor cells contain a lower areal density of synaptic vesicles in the Rs1h(tmgc1) retina. Examination of the synaptic interactions in the outer plexiform layer also revealed ectopic localization of photoreceptor cell presynaptic markers and elongation of neurites from postsynaptic neurons (bipolar and horizontal cells), which are observed in other mouse models with defective photoreceptor cell molecules. Consistent with these synaptic abnormalities, ERG analysis of young Rs1h(tmgc1) mice revealed attenuation of the b-wave with preservation of the a-wave. These results demonstrate that RS1H has functional significance in the morphology and function of the synapse between photoreceptors and second-order neurons. A developmental study from postnatal day (P) 15 through P19 showed that synaptic interactions form normally, and structural abnormalities occur after completion of synaptic formation suggesting that RS1H is important for the maintenance of this synaptic interaction. Thus, Rs1h(tmgc1) mice may serve as a new genetic model for human XLRS and other synaptic disorders.

The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses.

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.