Improving access to exome sequencing in a medically underserved population through the Texome Project.

PURPOSE: Genomic medicine can end diagnostic odysseys for patients with complex phenotypes; however, limitations in insurance coverage and other systemic barriers preclude individuals from accessing comprehensive genetics evaluation and testing. METHODS: The Texome Project is a 4-year study that reduces barriers to genomic testing for individuals from underserved and underrepresented populations. Participants with undiagnosed, rare diseases who have financial barriers to obtaining exome sequencing (ES) clinically are enrolled in the Texome Project. RESULTS: We highlight the Texome Project process and describe the outcomes of the first 60 ES results for study participants. Participants received a genetic evaluation, ES, and return of results at no cost. We summarize the psychosocial or medical implications of these genetic diagnoses. Thus far, ES provided molecular diagnoses for 18 out of 60 (30%) of Texome participants. Plus, in 11 out of 60 (18%) participants, a partial or probable diagnosis was identified. Overall, 5 participants had a change in medical management. CONCLUSION: To date, the Texome Project has recruited a racially, ethnically, and socioeconomically diverse cohort. The diagnostic rate and medical impact in this cohort support the need for expanded access to genetic testing and services. The Texome Project will continue reducing barriers to genomic care throughout the future study years.

Genetics and Pathogenesis of Parkinson's Syndrome.

Parkinson's disease (PD) is clinically, pathologically, and genetically heterogeneous, resisting distillation to a single, cohesive disorder. Instead, each affected individual develops a virtually unique form of Parkinson's syndrome. Clinical manifestations consist of variable motor and nonmotor features, and myriad overlaps are recognized with other neurodegenerative conditions. Although most commonly characterized by alpha-synuclein protein pathology throughout the central and peripheral nervous systems, the distribution varies and other pathologies commonly modify PD or trigger similar manifestations. Nearly all PD is genetically influenced. More than 100 genes or genetic loci have been identified, and most cases likely arise from interactions among many common and rare genetic variants. Despite its complex architecture, insights from experimental genetic dissection coalesce to reveal unifying biological themes, including synaptic, lysosomal, mitochondrial, andimmune-mediated mechanisms of pathogenesis. This emerging understanding of Parkinson's syndrome, coupled with advances in biomarkers and targeted therapies, presages successful precision medicine strategies.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Genome Sequencing in the Parkinson Disease Clinic.

Background and Objectives: Genetic variants affect both Parkinson disease (PD) risk and manifestations. Although genetic information is of potential interest to patients and clinicians, genetic testing is rarely performed during routine PD clinical care. The goal of this study was to examine interest in comprehensive genetic testing among patients with PD and document reactions to possible findings from genome sequencing in 2 academic movement disorder clinics. Methods: In 203 subjects with PD (age = 63 years, 67% male), genome sequencing was performed and filtered using a custom panel, including 49 genes associated with PD, parkinsonism, or related disorders, as well as a 90-variant PD genetic risk score. Based on the results, 231 patients (age = 67 years, 63% male) were surveyed on interest in genetic testing and responses to vignettes covering (1) familial risk of PD (LRRK2); (2) risk of PD dementia (GBA); (3) PD genetic risk score; and (4) secondary, medically actionable variants (BRCA1). Results: Genome sequencing revealed a LRRK2 variant in 3% and a GBA risk variant in 10% of our clinical sample. The genetic risk score was normally distributed, identifying 41 subjects with a high risk of PD. Medically actionable findings were discovered in 2 subjects (1%). In our survey, the majority (82%) responded that they would share a LRRK2 variant with relatives. Most registered unchanged or increased interest in testing when confronted with a potential risk for dementia or medically actionable findings, and most (75%) expressed interest in learning their PD genetic risk score. Discussion: Our results highlight broad interest in comprehensive genetic testing among patients with PD and may facilitate integration of genome sequencing in clinical practice.

A novel de novo intronic variant in ITPR1 causes Gillespie syndrome.

Gillespie syndrome (GLSP) is characterized by bilateral symmetric partial aplasia of the iris presenting as a fixed and large pupil, cerebellar hypoplasia with ataxia, congenital hypotonia, and varying levels of intellectual disability. GLSP is caused by either biallelic or heterozygous, dominant-negative, pathogenic variants in ITPR1. Here, we present a 5-year-old male with GLSP who was found to have a heterozygous, de novo intronic variant in ITPR1 (NM_001168272.1:c.5935-17G > A) through genome sequencing (GS). Sanger sequencing of cDNA from this individual's fibroblasts showed the retention of 15 nucleotides from intron 45, which is predicted to cause an in-frame insertion of five amino acids near the C-terminal transmembrane domain of ITPR1. In addition, qPCR and cDNA sequencing demonstrated reduced expression of both ITPR1 alleles in fibroblasts when compared to parental samples. Given the close proximity of the predicted in-frame amino acid insertion to the site of previously described heterozygous, de novo, dominant-negative, pathogenic variants in GLSP, we predict that this variant also has a dominant-negative effect on ITPR1 channel function. Overall, this is the first report of a de novo intronic variant causing GLSP, which emphasizes the utility of GS and cDNA studies for diagnosing patients with a clinical presentation of GLSP and negative clinical exome sequencing.

Integrated sequencing and array comparative genomic hybridization in familial Parkinson disease.

OBJECTIVE: To determine how single nucleotide variants (SNVs) and copy number variants (CNVs) contribute to molecular diagnosis in familial Parkinson disease (PD), we integrated exome sequencing (ES) and genome-wide array-based comparative genomic hybridization (aCGH) and further probed CNV structure to reveal mutational mechanisms. METHODS: We performed ES on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated ES and aCGH data for pathogenic SNVs and CNVs at Mendelian PD gene loci. We confirmed SNVs via Sanger sequencing and further characterized CNVs with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing. RESULTS: Using ES, we discovered individuals with known pathogenic SNVs in GBA (p.Glu365Lys, p.Thr408Met, p.Asn409Ser, and p.Leu483Pro) and LRRK2 (p.Arg1441Gly and p.Gly2019Ser). Two subjects were each double heterozygotes for variants in GBA and LRRK2. Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.Asn52Metfs*29, p.Thr240Met, p.Pro437Leu, and p.Trp453*) and likely disrupting CNVs at the PRKN locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors. CONCLUSIONS: Integrated ES and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PRKN CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

The expanding clinical phenotype of germline ABL1-associated congenital heart defects and skeletal malformations syndrome.

Congenital heart defects and skeletal malformations syndrome (CHDSKM) is a rare autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. CHDSKM is caused by germline mutations in ABL1. To date, three variants have been in association with CHDSKM. In this study, we describe three de novo missense variants, c.407C>T (p.Thr136Met), c.746C>T (p.Pro249Leu), and c.1573G>A (p.Val525Met), and one recurrent variant, c.1066G>A (p.Ala356Thr), in six patients, thereby expanding the phenotypic spectrum of CHDSKM to include hearing impairment, lipodystrophy-like features, renal hypoplasia, and distinct ocular abnormalities. Functional investigation of the three novel variants showed an increased ABL1 kinase activity. The cardiac findings in additional patients with p.Ala356Thr contribute to the accumulating evidence that patients carrying either one of the recurrent variants, p.Tyr245Cys and p.Ala356Thr, have a high incidence of cardiac abnormalities. The phenotypic expansion has implications for the clinical diagnosis of CHDSKM in patients with germline ABL1 variants.

De novo EIF2AK1 and EIF2AK2 Variants Are Associated with Developmental Delay, Leukoencephalopathy, and Neurologic Decompensation.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

De novo missense variant in the GTPase effector domain (GED) of DNM1L leads to static encephalopathy and seizures.

DNM1L encodes a GTPase of the dynamin superfamily, which plays a crucial role in mitochondrial and peroxisomal fission. Pathogenic variants affecting the middle domain and the GTPase domain of DNM1L have been implicated in encephalopathy because of defective mitochondrial and peroxisomal fission 1 (EMPF1, MIM #614388). Patients show variable phenotypes ranging from severe hypotonia leading to death in the neonatal period to developmental delay/regression, with or without seizures. Familial pathogenic variants in the GTPase domain have also been associated with isolated optic atrophy. We present a 27-yr-old woman with static encephalopathy, a history of seizures, and nystagmus, in whom a novel de novo heterozygous variant was detected in the GTPase effector domain (GED) of DNM1L (c.2072A>G, p.Tyr691Cys). Functional studies in Drosophila demonstrate large, abnormally distributed peroxisomes and mitochondria, an effect very similar to that of middle domain missense alleles observed in pediatric subjects with EMPF1. To our knowledge, not only is this the first report of a disease-causing variant in the GED domain in humans, but this is also the oldest living individual reported with EMPF1. Longitudinal data of this kind helps to expand our knowledge of the natural history of a growing list of DNM1L-related disorders.

Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease.

Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Use of Exome Sequencing for Infants in Intensive Care Units: Ascertainment of Severe Single-Gene Disorders and Effect on Medical Management.

Importance: While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective: To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants: Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures: Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results: The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance: Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.

NeuroChip, an updated version of the NeuroX genotyping platform to rapidly screen for variants associated with neurological diseases.

Genetics has proven to be a powerful approach in neurodegenerative diseases research, resulting in the identification of numerous causal and risk variants. Previously, we introduced the NeuroX Illumina genotyping array, a fast and efficient genotyping platform designed for the investigation of genetic variation in neurodegenerative diseases. Here, we present its updated version, named NeuroChip. The NeuroChip is a low-cost, custom-designed array containing a tagging variant backbone of about 306,670 variants complemented with a manually curated custom content comprised of 179,467 variants implicated in diverse neurological diseases, including Alzheimer's disease, Parkinson's disease, Lewy body dementia, amyotrophic lateral sclerosis, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, and multiple system atrophy. The tagging backbone was chosen because of the low cost and good genome-wide resolution; the custom content can be combined with other backbones, like population or drug development arrays. Using the NeuroChip, we can accurately identify rare variants and impute over 5.3 million common SNPs from the latest release of the Haplotype Reference Consortium. In summary, we describe the design and usage of the NeuroChip array and show its capability for detecting rare pathogenic variants in numerous neurodegenerative diseases. The NeuroChip has a more comprehensive and improved content, which makes it a reliable, high-throughput, cost-effective screening tool for genetic research and molecular diagnostics in neurodegenerative diseases.

Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation.

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

Missense variants in the middle domain of DNM1L in cases of infantile encephalopathy alter peroxisomes and mitochondria when assayed in Drosophila.

Defects in organelle dynamics underlie a number of human degenerative disorders, and whole exome sequencing (WES) is a powerful tool for studying genetic changes that affect the cellular machinery. WES may uncover variants of unknown significance (VUS) that require functional validation. Previously, a pathogenic de novo variant in the middle domain of DNM1L (p.A395D) was identified in a single patient with a lethal defect of mitochondrial and peroxisomal fission. We identified two additional patients with infantile encephalopathy and partially overlapping clinical features, each with a novel VUS in the middle domain of DNM1L (p.G350R and p.E379K). To evaluate pathogenicity, we generated transgenic Drosophila expressing wild-type or variant DNM1L. We find that human wild-type DNM1L rescues the lethality as well as specific phenotypes associated with the loss of Drp1 in Drosophila. Neither the p.A395D variant nor the novel variant p.G350R rescue lethality or other phenotypes. Moreover, overexpression of p.A395D and p.G350R in Drosophila neurons, salivary gland and muscle strikingly altered peroxisomal and mitochondrial morphology. In contrast, the other novel variant (p.E379K) rescued lethality and did not affect organelle morphology, although it was associated with a subtle mitochondrial trafficking defect in an in vivo assay. Interestingly, the patient with the p.E379K variant also has a de novo VUS in pyruvate dehydrogenase 1 (PDHA1) affecting the same amino acid (G150) as another case of PDHA1 deficiency suggesting the PDHA1 variant may be pathogenic. In summary, detailed clinical evaluation and WES with functional studies in Drosophila can distinguish different functional consequences of newly-described DNM1L alleles.

Whole-Exome Sequencing in Familial Parkinson Disease.

IMPORTANCE: Parkinson disease (PD) is a progressive neurodegenerative disease for which susceptibility is linked to genetic and environmental risk factors. OBJECTIVE: To identify genetic variants contributing to disease risk in familial PD. DESIGN, SETTING, AND PARTICIPANTS: A 2-stage study design that included a discovery cohort of families with PD and a replication cohort of familial probands was used. In the discovery cohort, rare exonic variants that segregated in multiple affected individuals in a family and were predicted to be conserved or damaging were retained. Genes with retained variants were prioritized if expressed in the brain and located within PD-relevant pathways. Genes in which prioritized variants were observed in at least 4 families were selected as candidate genes for replication in the replication cohort. The setting was among individuals with familial PD enrolled from academic movement disorder specialty clinics across the United States. All participants had a family history of PD. MAIN OUTCOMES AND MEASURES: Identification of genes containing rare, likely deleterious, genetic variants in individuals with familial PD using a 2-stage exome sequencing study design. RESULTS: The 93 individuals from 32 families in the discovery cohort (49.5% [46 of 93] female) had a mean (SD) age at onset of 61.8 (10.0) years. The 49 individuals with familial PD in the replication cohort (32.6% [16 of 49] female) had a mean (SD) age at onset of 50.1 (15.7) years. Discovery cohort recruitment dates were 1999 to 2009, and replication cohort recruitment dates were 2003 to 2014. Data analysis dates were 2011 to 2015. Three genes containing a total of 13 rare and potentially damaging variants were prioritized in the discovery cohort. Two of these genes (TNK2 and TNR) also had rare variants that were predicted to be damaging in the replication cohort. All 9 variants identified in the 2 replicated genes in 12 families across the discovery and replication cohorts were confirmed via Sanger sequencing. CONCLUSIONS AND RELEVANCE: TNK2 and TNR harbored rare, likely deleterious, variants in individuals having familial PD, with similar findings in an independent cohort. To our knowledge, these genes have not been previously associated with PD, although they have been linked to critical neuronal functions. Further studies are required to confirm a potential role for these genes in the pathogenesis of PD.

Molecular basis of the interactions of the Nogo-66 receptor and its homolog NgR2 with myelin-associated glycoprotein: development of NgROMNI-Fc, a novel antagonist of CNS myelin inhibition.

Myelin-associated glycoprotein (MAG) is a sialic acid-binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and uses neuronal cell-type-specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high affinity and in a sialic acid-dependent manner to the Nogo-66 receptor-1 (NgR1) and its homolog NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp, and MAG. Deletion of disulfide loop Cys(309)-Cys(336) of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR(OMNI)) in which Cys(309)-Cys(336) is deleted and followed by a 13 aa MAG-binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared with wild-type NgR1 or NgR2. Soluble NgR(OMNI) (NgR(OMNI)-Fc) binds strongly to membrane-bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR(OMNI)-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.

Mechanisms of CNS myelin inhibition: evidence for distinct and neuronal cell type specific receptor systems.

Following injury to the adult mammalian central nervous system, regenerative growth of severed axons is very limited. The lack of neuronal repair is often associated with significant functional deficits, and depending on the severity of injury, may result in permanent paralysis distal to the site of injury. A detailed understanding of the molecular mechanisms that limit neuronal growth in the injured spinal cord is an important step toward the development of specific strategies aimed at restoring functional connectivity lost as a consequence of injury. While rapid progress is being made in defining the molecular identity of CNS growth inhibitory constituents, comparatively little is known about their receptors and downstream signaling mechanisms. Emerging new evidence suggests that the mechanisms for myelin inhibition are likely to be complex, involving multiple and distinct receptor systems that may operate in a redundant manner. Furthermore, the relative contribution of a specific ligand-receptor system to bring about growth inhibition may greatly vary among different neuronal cell types. Myelin-associated glycoprotein (MAG), for example, employs different mechanisms to inhibit neurite outgrowth of cerebellar, sensory, and retinal ganglion neurons in vitro. Nogo-A harbors distinct growth inhibitory regions, which employ different signaling mechanisms. The Nogo-66 receptor 1 (NgR1), a shared ligand binding component in a receptor complex for Nogo-66, MAG, and OMgp, participates in neuronal growth cone collapse to acutely presented myelin inhibitors, but is dispensable for longitudinal neurite outgrowth inhibition on substrate-bound Nogo-66, MAG, OMgp, or crude CNS myelin in vitro. Consistent with the idea of cell-type specific mechanisms for myelin inhibition, different types of CNS neurons possess very different regenerative capacities and respond differently to experimental treatment strategies in vivo. We speculate that differences in regenerative axonal growth among different fiber systems are a reflection of their intrinsic ability to elongate axons and their distinct cell surface receptor profiles to respond to the growth inhibitory extracellular milieu. The existence of cell type specific mechanisms to impair regenerative axonal growth in the CNS may have important implications for the development of treatment strategies. Depending on the fiber tract injured, different ligand-receptor systems may need to be targeted in order to elicit robust and long-distance regenerative axonal growth.