Robert N. Jinks

Genetic Mapping and Exome Sequencing Identify Variants Associated with Five Novel Diseases

The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data.

A homozygous missense mutation in HERC2 associated with global developmental delay and autism spectrum disorder

We studied a unique phenotype of cognitive delay, autistic behavior, and gait instability segregating in three separate sibships. We initiated genome‐wide mapping in two sibships using Affymetrix 10K SNP Mapping Arrays and identified a homozygous 8.2 Mb region on chromosome 15 common to five affected children. We used exome sequencing of two affected children to assess coding sequence variants within the mapped interval. Four novel homozygous exome variants were shared between the two patients; however, only two variants localized to the mapped interval on chromosome 15. A third sibship in an Ohio Amish deme narrowed the mapped interval to 2.6 Mb and excluded one of the two novel homozygous exome variants. The remaining variant, a missense change in HERC2 (c.1781C>T, p.Pro594Leu), occurs in a highly conserved proline residue within an RCC1‐like functional domain. Functional studies of truncated HERC2 in adherent retinal pigment epithelium cells suggest that the p.Pro594Leu variant induces protein aggregation and leads to decreased HERC2 abundance. The phenotypic correlation with the mouse Herc1 and Herc2 mutants as well as the phenotypic overlap with Angelman syndrome provide further evidence that pathogenic changes in HERC2 are associated with nonsyndromic intellectual disability, autism, and gait disturbance. Hum Mutat 33:1639–1646, 2012.

Adaptive visual metamorphosis in a deep-sea hydrothermal vent crab

Hydrothermal vents along the mid-ocean ridges host ephemeral ecosystems of diverse endemic fauna including several crustacean species, some of which undergo planktonic development as larvae up to 1,000 m above and 100 km away from the vents. Little is known about the role of vision in the life history of vent fauna. Here we report that planktonic zoea larvae of the vent crab Bythograea thermydron possess image-forming compound eyes with a visual pigment sensitive to the blue light of mesopelagic waters. As they metamorphose and begin to descend to and settle at the vents, they lose their image-forming optics and develop high-sensitivity naked-retina eyes. The spectral absorbance of the visual pigment in these eyes shifts towards longer wavelengths from larva to postlarva to adult. This progressive visual metamorphosis trades imaging for increased sensitivity, and changes spectral sensitivity from the blue wavelengths of the larval environment towards the dim, longer wavelengths produced in the deeper bathypelagic vent environment of the adults. As hydrothermal vents produce light, vision may supplement thermal and chemical senses to orient postlarval settlement at vent sites.

Sulfide as a Chemical Stimulus for Deep-Sea Hydrothermal Vent Shrimp

Organisms dependent on deep-sea hydrothermal vents for their existence face extinction when their vents expire, unless they can establish populations on neighboring vents or on new vent sites. Propagules, including larvae and motile adults, are readily dispersed broadly by seafloor currents, but how they recognize active hydrothermal sites is problematical. Compelling evidence that vent organisms can find and colonize hydrothermal sites has been provided by a series of observations on the East Pacific Rise (1). New hydrothermal vents created there following a volcanic eruption on the seafloor in March 1991 were colonized by sessile invertebrates in less than one year. On the Mid-Atlantic Ridge, shrimp that normally cluster on sulfide surfaces have been observed to swim directly back to the surfaces when displaced from them. How do vent animals locate new or existing vents? Passive transport by currents (2) or active swimming without guidance by some physical cue is not likely to result in success (3). Chemicals present in hydrothermal fluids have been proposed as attractants. We provide the first evidence of a chemosensory response in a vent invertebrate to sulfides, which are prevalent in vent fluids and provide the energy,for chemosynthetic primary production at vents.

A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ2 = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Retinal Anatomy of a New Species of Bresiliid Shrimp From a Hydrothermal Vent Field on the Mid-Atlantic Ridge

A new species of shrimp (Rimicaris sp.) was recently collected from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge. Until the discovery in 1989 that the deep-sea, hydrothermal vent species, Rimicaris exoculata, possessed a hypertrophied dorsal eye, everyone believed that animals recovered from vent environments were blind. Like R. exoculata, Rimicaris sp., a small orange bresiliid shrimp, has an enlarged dorsal eye specialized for detecting light in a very dim environment instead of the expected compound eye. The individual lenses characteristic of a compound eye adapted for imaging have been replaced in Rimicaris sp. by a smooth cornea underlain by a massive array of photosensitive membrane. The number of ommatidia in this species is about the same as in shrimp species that live at the surface; however, the photoreceptors are larger in the deep-sea species and the shape of the photoreceptors is markedly different. The light-sensitive region of the photoreceptor is much larger than those of other shrimp and the rest of the receptor is much smaller than normal. All screening pigment has moved out of the path of incident light to a position below the retina, and the reflecting pigment cells have adapted to form a bright white diffusing screen between and behind the photoreceptors. The ultrastructure of the microvillar array comprising the rhabdom is typical for decapod crustaceans; however, there is a much greater volume density of rhabdom (80% to 85%) than normal. There is no ultrastructural evidence for cyclic rhabdom shedding or renewal. Rimicaris sp. has apparently adapted its visual system to detect the very dim light emitted from the throats of the black smoker chimneys around which it lives.

Limulus opsins: Diurnal regulation of expression

Much has been learned from studies of Limulus photoreceptors about the role of the circadian clock and light in the removal of photosensitive membrane. However, little is known in this animal about mechanisms regulating photosensitive membrane renewal, including the synthesis of proteins in, and associated with, the photosensitive membrane. To begin to understand renewal, this study examines diurnal changes in the levels of mRNAs encoding opsin, the integral membrane protein component of visual pigment, and the relative roles of light and the circadian clock in producing these changes. We show that at least two distinct opsin genes encoding very similar proteins are expressed in both the lateral and ventral eyes, and that during the day and night in the lateral eye, the average level of mRNA encoding opsinl is consistently higher than that encoding opsin2. Northern blot assays showed further that total opsin mRNA in the lateral eyes of animals maintained under natural illumination increases during the afternoon (9 & 12 h after sunrise) in the light and falls at night in the dark. This diurnal change occurs whether or not the eyes receive input from the circadian clock, but it is eliminated in eyes maintained in the dark. Thus, it is regulated by light and darkness, not by the circadian clock, with light stimulating an increase in opsin mRNA levels. The rise in opsin mRNA levels observed under natural illumination was seasonal; it occurred during the summer but not the spring and fall. However, a significant increase in opsin mRNA levels could be achieved in the fall by exposing lateral eyes to 3 h of natural illumination followed by 9 h of artificial light. The diurnal regulation of opsin mRNA levels contrasts sharply with the circadian regulation of visual arrestin mRNA levels (Battelle et al., 2000). Thus, in Limulus, distinctly different mechanisms regulate the levels of mRNA encoding two proteins critical for the photoresponse.

Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozygous protein-truncating mutations of WDR73

Galloway-Mowat syndrome (GMS) is a neurodevelopmental disorder characterized by microcephaly, cerebellar hypoplasia, nephrosis, and profound intellectual disability. Jinks et al. extend the GMS spectrum by identifying a novel nephrocerebellar syndrome with selective striatal cholinergic interneuron loss and complete lateral geniculate nucleus delamination, caused by a frameshift mutation in WDR73.

Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant

Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thickening and reduced gyration. Here we describe a “thin” lissencephaly (TLIS) variant characterized by megalencephaly, frontal predominant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-β-induced dendritic spine collapse and neuronal apoptosis, suggesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD’s ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mechanism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability.

Protein kinase C-induced disorganization and endocytosis of photosensitive membrane in Limulus ventral photoreceptors.

Protein kinase C (PKC) desensitizes the light response in photoreceptors from the ventral optic nerve of the horseshoe crab Limulus. Photoisomerization of Limulus rhodopsin leads to phosphoinositide hydrolysis, resulting in the production of inositol trisphosphate and diacylglycerol (DAG). Inositol trisphosphate mobilizes intracellular stores of Ca2+, resulting in photoreceptor excitation in Limulus, while DAG may activate PKC. We investigated whether PKC‐mediated desensitization of the photoresponse is accompanied by ultrastructural changes in the rhodopsin‐bearing photosensitive membrane (rhabdom) in Limulus ventral photoreceptors. PKC activation by (–)‐indolactam V in darkness induces disorganization and swelling of the rhodopsin‐containing microvilli and endocytosis of rhabdomeral membrane. The effects of (–)‐indolactam V on dark‐adapted photoreceptor ultrastructure are reversible, are stereospecific, are blocked by coapplication of PKC inhibitors, and closely match those induced by continuous, bright light. Rhabdom disorganization and endocytosis via PKC activation may, therefore, contribute to desensitization of the light‐adapted photoreceptor. J. Comp. Neurol. 442:217–225, 2002.