Total anomalous pulmonary venous connection (TAPVC) is a potentially lethal congenital disorder that occurs when the pulmonary veins do not connect normally to the left atrium, allowing mixing of pulmonary and systemic blood1. In contrast to the extensive knowledge of arterial vascular patterning, little is known about the patterning of veins. Here we show that the secreted guidance molecule semaphorin 3d (Sema3d) is crucial for the normal patterning of pulmonary veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand (MES), the precursor of the common pulmonary vein, does not form at the proper location on the dorsal surface of the embryonic common atrium2,3. However, we found that TAPVC occurs in Sema3d mutant mice despite normal formation of the MES. In these embryos, the maturing pulmonary venous plexus does not anastomose uniquely with the properly formed MES. In the absence of Sema3d, endothelial tubes form in a region that is normally avascular, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary. Sequencing of SEMA3D in individuals with anomalous pulmonary veins identified a phenylalanine-to-leucine substitution that adversely affects SEMA3D function. These results identify Sema3d as a crucial pulmonary venous patterning cue and provide experimental evidence for an alternate developmental model to explain abnormal pulmonary venous connections.

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Hearing impairment affects 1 in 650 newborns, making it the most common congenital sensory impairment. Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing impairment cases. Mutations in GJB2 account for a significant number of ARNSHI (and up to 50% of documented recessive (e.g. more than 1 affected sibling) hearing impairment in some populations). Mutations in the GJB2 gene are amongst the most common causes of hearing impairment in populations of various ethnic backgrounds. Two mutations of this gene, 35delG and 167delT, account for the majority of reported mutations in Caucasian populations, especially those of Mediterranean and Ashkenazi Jewish background. The 235delC mutation is most prevalent in East Asian populations. Some mutations are of less well characterized significance. The V37I missense mutation, common in Asian populations, was initially described as a polymorphism and later as a potentially pathogenic mutation. We report here on 15 unrelated individuals with ARNSHI and homozygosity for the V37I GJB2 missense mutation. Nine individuals are of Chinese ancestry, two are of unspecified Asian descent, one is of Japanese descent, one individual is of Vietnamese ancestry, one of Philippine background and one of Italian and Cuban/Caucasian background. Homozygosity for the V37I GJB2 mutation may be a more common pathogenic missense mutation in Asian populations, resulting in mild to moderate sensorineural hearing impairment. We report a presumed haplotype block specific to East Asian individuals with the V37I mutation encompassing the GJB2 gene that may account for the high prevalence in East Asian populations.

This report describes an algorithm developed to predict the pathogenicity of copy number variants (CNVs) in large sample cohorts. CNVs (genomic deletions and duplications) are found in healthy individuals and in individuals with genetic diagnoses, and differentiation of these two classes of CNVs can be challenging and usually requires extensive manual curation. We have developed PECONPI, an algorithm to assess the pathogenicity of CNVs based on gene content and CNV frequency. This software was applied to a large cohort of patients with genetically heterogeneous non-syndromic hearing loss to score and rank each CNV based on its relative pathogenicity. Of 636 individuals tested, we identified the likely underlying etiology of the hearing loss in 14 (2%) of the patients (1 with a homozygous deletion, 7 with a deletion of a known hearing loss gene and a point mutation on the trans allele and 6 with a deletion larger than 1 Mb). We also identified two probands with smaller deletions encompassing genes that may be functionally related to their hearing loss. The ability of PECONPI to determine the pathogenicity of CNVs was tested on a second genetically heterogenous cohort with congenital heart defects (CHDs). It successfully identified a likely etiology in 6 of 355 individuals (2%). We believe this tool is useful for researchers with large genetically heterogeneous cohorts to help identify known pathogenic causes and novel disease genes.