Long‐read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel–Gruber syndrome

Abstract

The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule longread sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

A Novel Homozygous Variant of TMEM231 in a Case With Hypoplasia of the Cerebellar Vermis and Polydactyly

“…MKS is a much more deadly perinatal syndrome. As the severe part of the ciliopathy phenotypic spectrum, MKS-affected individuals present with polycystic kidneys, occipital encephalocele, and polydactyly ( 13 ). To date, mutations in more than 27 genes are associated with JBTS, and pathogenic variants in at least 17 genes have been identified in MKS patients.…”

“…To date, mutations in more than 27 genes are associated with JBTS, and pathogenic variants in at least 17 genes have been identified in MKS patients. Importantly, mutations in 12 of these genes, including TMEM231, TMEM67 , and TEME237, are reported to cause both conditions ( 13 , 14 ). As JBTS and MKS share overlapping features and are genetically heterogeneous, some laboratories routinely deploy a gene panel diagnostic approach that examines all JBTS- or MKS- associated disease genes, irrespective of which of the two diagnostic categories the clinical features suggest ( 13 ).…”

Molecular characterization of pathogenic OTOA gene conversions in hearing loss patients

“…Gene conversion, defined as the replacement of a locus in the genome by a paralogous sequence, is a mechanism known to generate pathogenic alleles (Casola et al, 2012; Chen et al, 2007). For instance, a pathogenic converted allele has been described for the TMEM231 gene, causing autosomal recessive Joubert and Meckel‐Gruber syndromes (Maglic et al, 2016; Watson et al, 2020). Other examples include a benign conversion of 88–351 nucleotides of BRCA1 intron 2 (Tessereau et al, 2015) mediated by the same breakpoint sites leading to a 37 kbp‐long deletion of exon 1 and 2 of BRCA1 .…”

Overcoming the pitfalls of NGS-based molecular diagnosis of Shwachman-Diamond syndrome

“…Disentangling the disease-causing variants through short NGS reads is frequently limited since SBDS shares 97% identity with SBDSP1 [21][22][23] . Yamada et al reported two SDS patients with compound heterozygous mutations in the SBDS gene (c.258+2T>C on one allele and c.183_184delinsCT together with c.201A>G on the other), validated by Sanger sequencing.…”