The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

Background Trisomy 13 occurs in 1/10 000–20 000 live births, and mosaicism accounts for 5% of these cases. Phenotype and outcome of mosaic trisomy 13 are variable and poorly understood. Microsatellite analyses of trisomy 13 have indicated the high incidence of maternal meiotic origin and reduced recombination, but no study has focused on mosaic trisomy 13 in live born patients. Methods and results Single-nucleotide polymorphism (SNP) array, fluorescence in situ hybridisation and bioinformatics analyses were performed in three cases of mosaic trisomy 13. Two cases of complete mosaic trisomy 13 originated from meiosis I non-disjunction followed by trisomic rescue; one had crossovers resulting in segmental uniparental disomy in the disomic line, and one had no crossover. Mosaicism for partial trisomy 13 in the third complex case either arose from meiosis II non-disjunction without crossover or in early mitosis followed by anaphase lags. The extra chromosome 13 was maternal in origin in all three cases. Mosaicism percentage calculated from B allele frequencies ranged from 30 to 50. Conclusions Genotypes and copy number information provided by SNP array allow determination of parental origin and uniparental disomy status and direct quantification of mosaicism. Such information may lead to a better understanding of mechanisms underlying mosaic aneuploidies and the observed phenotypic variability and better prediction of recurrent risk.

A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T>C (p.Phe83Leu) and c.560G>A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

Ollier disease (OD) and Maffucci Syndrome (MS) are rare disorders characterized by multiple enchondromas, commonly causing bone deformities, limb length discrepancies, and pathological fractures. MS is distinguished from OD by the development of vascular anomalies. Both disorders are cancer predisposition syndromes with malignancies developing in ~50% of the individuals with OD or MS. Somatic gain-of-function variants in IDH1 and IDH2 have been described in the enchondromas, vascular anomalies and chondrosarcomas of approximately 80% of the individuals with OD and MS. To date, however, no investigation of germline causative variants for these diseases has been comprehensively performed. To search for germline causative variants, we performed whole exome sequencing or whole genome sequencing of blood or saliva DNA in 94 unrelated probands (68 trios). We found that 7 had rare germline missense variants in HIF1A, 6 had rare germline missense variants in VHL, and 3 had IDH1 variants including 2 with mosaic IDH1-p.Arg132His variant. A burden analysis using 94 probands assigned as cases and 2,054 unrelated individuals presenting no OD- or MS-related features as controls, found that variants in HIF1A, VHL, and IDH1 were all significantly enriched in cases compared to controls. To further investigate the role of HIF-1 pathway in the pathogenesis of OD and MS, we performed RNA sequencing of fibroblasts from 4 probands with OD or MS at normoxia and at hypoxia. When cultured in hypoxic conditions, both proband and control cells showed altered expression of a subset of HIF-1 regulated genes. However, the set of differentially expressed genes in proband fibroblasts included a significantly reduced number of HIF-1 regulated genes compared to controls. Our findings suggest that germline or early post-zygotic variants identified in HIF1A, VHL, and IDH1 in probands with OD and MS underlie the development of the phenotypic abnormalities in a subset of individuals with OD and MS, but extensive functional studies are needed to further confirm it.

Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) may be misdiagnosed as primary congenital glaucoma (PCG) due to similar clinical phenotypes during early infancy. In this study, we identified a family with CHED2, which was previously misdiagnosed as having PCG, and followed up for 9 years. Linkage analysis was first completed in eight PCG-affected families, followed by whole-exome sequencing (WES) in family PKGM3. The following in silico tools were used to predict the pathogenic effects of identified variants: I-Mutant 2.0, SIFT, Polyphen-2, PROVEAN, mutation taster and PhD-SNP. After detecting an SLC4A11 variant in one family, detailed ophthalmic examinations were performed again to confirm the diagnosis. Six out of eight families had CYP1B1 gene variants responsible for PCG. However, in family PKGM3, no variants in the known PCG genes were identified. WES identified a homozygous missense variant c.2024A>C, p.(Glu675Ala) in SLC4A11. Based on the WES findings, the affected individuals underwent detailed ophthalmic examinations and were re-diagnosed with CHED2 leading to secondary glaucoma. Our results expand the genetic spectrum of CHED2. This is the first report from Pakistan of a Glu675Ala variant with CHED2 leading to secondary glaucoma. The p.Glu675Ala variant is likely a founder mutation in the Pakistani population. Our findings suggest that genome-wide neonatal screening is worthwhile to avoid the misdiagnosis of phenotypically similar diseases such as CHED2 and PCG.

Mutations in the gene kyphoscoliosis peptidase (KY) are known to cause myofibrillar myopathy-7 and hereditary spastic paraplegia. We investigated the genetic cause of a complex neurological phenotype in a consanguineous Pakistani family with four affected members, manifesting lower limb spasticity and weakness, toe walking, pes equinovarus, and a speech disorder. Genome-wide linkage analysis with microsatellite markers delineated chromosome 3q22.2-q24 harboring the disease gene. Whole exome sequencing was performed for two subjects, identifying a homozygous 14-bp frameshift deletion NM_178554.6:c.842_855del;p.(Val281GlyfsTer18) in KY. The variant segregated with the phenotype and was absent from public databases and 100 ethnically matched controls. We confirm a novel homozygous KY variant causing a complex neurological phenotype in this family. A review of previously reported KY variants suggests that variants in this gene can cause a spectrum of neurological phenotypes.