Discovery and Functional Annotation of SIX6 Variants in Primary Open-Angle Glaucoma

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Multidisciplinary approaches for elucidating genetics and molecular pathogenesis of urinary tract malformations

Advances in clinical diagnostics and molecular tools have improved our understanding of the genetically heterogeneous causes underlying congenital anomalies of kidney and urinary tract (CAKUT). However, despite a sharp incline of CAKUT reports in the literature within the past 2 decades, there remains a plateau in the genetic diagnostic yield that is disproportionate to the accelerated ability to generate robust genome-wide data. Explanations for this observation include (i) diverse inheritance patterns with incomplete penetrance and variable expressivity, (ii) rarity of single-gene drivers such that large sample sizes are required to meet the burden of proof, and (iii) multigene interactions that might produce either intra- (e.g., copy number variants) or inter- (e.g., effects in trans) locus effects. These challenges present an opportunity for the community to implement innovative genetic and molecular avenues to explain the missing heritability and to better elucidate the mechanisms that underscore CAKUT. Here, we review recent multidisciplinary approaches at the intersection of genetics, genomics, in vivo modeling, and in vitro systems toward refining a blueprint for overcoming the diagnostic hurdles that are pervasive in urinary tract malformation cohorts. These approaches will not only benefit clinical management by reducing age at molecular diagnosis and prompting early evaluation for comorbid features but will also serve as a springboard for therapeutic development

Putative digenic inheritance of heterozygous <i>RP1L1</i> and <i>C2orf71</i> null mutations in syndromic retinal dystrophy

<p><i>Background</i>: Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration and can occur in non-syndromic and syndromic forms. Syndromic RP is accompanied by other symptoms such as intellectual disability, hearing loss, or congenital abnormalities. Both forms are known to exhibit complex genetic interactions that can modulate the penetrance and expressivity of the phenotype.</p> <p><i>Materials and methods</i>: In an individual with atypical RP, hearing loss, ataxia and cerebellar atrophy, whole exome sequencing was performed. The candidate pathogenic variants were tested by developing an <i>in vivo</i> zebrafish model and assaying for retinal and cerebellar integrity.</p> <p><i>Results</i>: Exome sequencing revealed a complex heterozygous protein-truncating mutation in <i>RP1L1</i>, p.[(Lys111Glnfs*27; Gln2373*)], and a heterozygous nonsense mutation in <i>C2orf71</i>, p.(Ser512*). Mutations in both genes have previously been implicated in autosomal recessive non-syndromic RP, raising the possibility of a digenic model in this family. Functional testing in a zebrafish model for two key phenotypes of the affected person showed that the combinatorial suppression of <i>rp1l1</i> and <i>c2orf71l</i> induced discrete pathology in terms of reduction of eye size with concomitant loss of rhodopsin in the photoreceptors, and disorganization of the cerebellum.</p> <p><i>Conclusions</i>: We propose that the combination of heterozygous loss-of-function mutations in these genes drives syndromic retinal dystrophy, likely through the genetic interaction of at least two loci. Haploinsufficiency at each of these loci is insufficient to induce overt pathology.</p

<i>In vivo</i> zebrafish morpholino complementation assay showing the effect of <i>SIX6</i> nonsynonymous variants.

<p>Zebrafish embryos were microinjected with a translation blocking morpholino designed to target <i>six6a</i>. Total eye size (µm²) was measured 3 days post fertilization. Compared to the uninjected controls, morphants showed a significant reduction in eye size. Zebrafish were co-injected with the morpholino and a human <i>SIX6</i> allele (Glu93Gln, Glu129Lys, Asn141His, Leu205Arg, Thr212Met, or Ser242IIe). Results of each allele were compared to the <i>SIX6</i> non-risk allele (Ref). P-values are provided below the mean of each treatment.</p

Functional evaluation of <i>SIX6</i> variants on the volume of the optic nerve.

<p>Representative whole mount images of acetylated-tubulin expression in the heads of zebrafish embryos injected with a control or <i>six6a</i> morpholino, rescued by co-injection with human non-risk SIX6 transcript or a transcript containing the Leu205Arg hypomorphic variant (A). Acetylated-tubulin staining is restricted primarily to axon tracts and can be used to visualize the optic nerve. Relative to the control morphants, volumetric regions of interest (ROI) along the optic nerve in <i>six6a</i> morphants were reduced significantly. Co-injection of human variants revealed a hypomorphic (Leu205Arg, Asn141His) or benign (Glu93Gln) role of the variants on the optic nerve (B). Sample size for all injection paradigms ranged from 7–9 and p-values are plotted for each comparison (*** p<0.001; ** p<0.01). No significant changes in the volume of other axonal tracts in the head (marked by an asterisk) were detected. Standard error of the mean is shown and white scale bars = 20 um.</p