Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously ...undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions.
We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGUeye) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants.
Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.Arg26Glnx1, p.Gly36Valx1, p.Arg38Trpx2, p.Arg38Glnx1, p.Gly51Argx2, p.Ser54Argx2, p.Asn124Lysx5) and one the homeodomain (p.Asn260Tyrx1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease.
Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Treacher Collins syndrome (TCS) is a rare autosomal dominant mandibulofacial dysostosis, with a prevalence of 0.2–1/10,000. Features include bilateral and symmetrical malar and mandibular hypoplasia ...and facial abnormalities due to abnormal neural crest cell (NCC) migration and differentiation. To date, three genes have been identified: TCOF1, POLR1C, and POLR1D. Despite a large number of patients with a molecular diagnosis, some remain without a known genetic anomaly.
We performed exome sequencing for four individuals with TCS but who were negative for pathogenic variants in the known causative genes. The effect of the pathogenic variants was investigated in zebrafish.
We identified three novel pathogenic variants in POLR1B. Knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype mimicking TCS that was associated with altered ribosomal gene expression, massive p53-associated cellular apoptosis in the neuroepithelium, and reduced number of NCC derivatives.
Pathogenic variants in the RNA polymerase I subunit POLR1B might induce massive p53-dependent apoptosis in a restricted neuroepithelium area, altering NCC migration and causing cranioskeletal malformations. We identify POLR1B as a new causative gene responsible for a novel TCS syndrome (TCS4) and establish a novel experimental model in zebrafish to study POLR1B-related TCS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify ...the missing heritability.
Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.
In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.
Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, ...migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The ...genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region ...containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ...ubiquity of a >4-fold susceptibility from a C→T allele (rs2435357: p = 3.9 × 10−43 in European ancestry; p = 1.1 × 10−21 in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%–30%/47% and case frequency of 54%–62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 × 10−5) and familiality (p = 6.2 × 10−4). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a “cellular-recessive” genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Objective
Conventional genetic tests (quantitative fluorescent‐PCR QF‐PCR and single nucleotide polymorphism‐array) only diagnose ~40% of fetuses showing ultrasound abnormalities. Rapid exome ...sequencing (rES) may improve this diagnostic yield, but includes challenges such as uncertainties in fetal phenotyping, variant interpretation, incidental unsolicited findings, and rapid turnaround times. In this study, we implemented rES in prenatal care to increase diagnostic yield.
Methods
We prospectively studied 55 fetuses. Inclusion criteria were: (a) two or more independent major fetal anomalies, (b) hydrops fetalis or bilateral renal cysts alone, or (c) one major fetal anomaly and a first‐degree relative with the same anomaly. In addition to conventional genetic tests, we performed trio rES analysis using a custom virtual gene panel of ~3850 Online Mendelian Inheritance in Man (OMIM) genes.
Results
We established a genetic rES‐based diagnosis in 8 out of 23 fetuses (35%) without QF‐PCR or array abnormalities. Diagnoses included MIRAGE (SAMD9), Zellweger (PEX1), Walker‐Warburg (POMGNT1), Noonan (PTNP11), Kabuki (KMT2D), and CHARGE (CHD7) syndrome and two cases of Osteogenesis Imperfecta type 2 (COL1A1). In six cases, rES diagnosis aided perinatal management. The median turnaround time was 14 (range 8‐20) days.
Conclusion
Implementing rES as a routine test in the prenatal setting is challenging but technically feasible, with a promising diagnostic yield and significant clinical relevance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with ...mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.
We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.
In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.
We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.
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To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants.
The coding and ...noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospectively. Two asymptomatic siblings carrying the same ABCA4 variants as their probands were clinically examined. The penetrance of p.Asn1868Ile was calculated using allele frequency data of ABCA4 variants in non-Finnish European individuals.
The p.Asn1868Ile variant was found in cis with known variants in 14/67 probands. In 27/67 probands, we identified p.Asn1868Ile without additional variants in cis, in combination with known, mainly severe ABCA4 variants. In 23/27 probands, the trans configuration was established. Among 27 probands and 6/7 STGD1 siblings carrying p.Asn1868Ile, 42% manifested late-onset disease (>44 years). We additionally identified four asymptomatic relatives carrying a combination of a severe variant and p.Asn1868Ile; ophthalmologic examination in two persons did not reveal STGD1. Based on ABCA4 allele frequency data, we conservatively estimated the penetrance of p.Asn1868Ile, when present in trans with a severe variant, to be below 5%.
A significant fraction of genetically unexplained STGD1 cases carries p.Asn1868Ile as a second variant. Our findings suggest exceptional differences in disease expression or even nonpenetrance of this ABCA4 variant, pointing toward an important role for genetic or environmental modifiers in STGD1.
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