TMEM231Gene Conversion Associated with Joubert and Meckel-Gruber Syndromes in the Same Family

Abstract

Joubert and Meckel-Gruber syndromes (JS and MGS) are ciliopathies with overlapping features. JS patients manifest the "molar tooth sign" on brain imaging and variable eye, kidney, and liver disease. MGS presents with polycystic kidneys, occipital encephalocele, and polydactyly; it is typically perinatally fatal. Both syndromes are genetically heterogeneous; some genes cause either syndrome. Here, we report two brothers married to unrelated women. The first brother had three daughters with JS and a son with polycystic kidneys who died at birth. The second brother's wife had a fetal demise due to MGS. Whole exome sequencing identified TMEM231 NM_001077416.2: c.784G>A; p.(Asp262Asn) in all children and the wife of the first brother; the second brother's wife had a c.406T>G;p.(Trp136Gly) change. In-depth analysis uncovered a rare gene conversion event in TMEM231, leading to loss of exon 4, in all the affected children of first brother. We believe that the combination of this gene conversion with different missense mutations led to a spectrum of phenotypes that span JS and MGS.

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

“…The expected exon 4 deletion could not be validated by this analysis, but we instead observed four clustered substitution variants (c.742‐1G>A, c.742A>G, c.747C>T, and c.752T>C) that were present in the long‐read, but not the short‐read, datasets (Figure ). The same clustered variants have previously been observed and attributed to a gene conversion event occurring between TMEM231 and a pseudogene located approximately 40 kb downstream (centromeric) from the functional gene (Maglic et al, ). It, therefore, appeared that during alignment, reassignment of short reads containing pseudogene‐derived variants, from TMEM231 to the more closely‐matching pseudogene locus, was the explanation for the apparent loss of read‐depth at exon 4.…”

“…We confirmed the presence of the conversion event by Sanger sequencing a 1187‐bp amplicon derived from the gene‐specific intron 3/exon 4 loci and showed that it was paternally inherited (Figure a). Two flanking pseudogene‐derived variants (c.742‐49G>A and c.823+4A>G) reported by Maglic et al () were not detected on the converted allele in our patient (data not shown); suggesting that the allele we have observed reflects a recurrent gene conversion event, rather than an ancestral founder allele.…”

“…To confirm and genotype the TMEM231 intron 3/exon 4 gene conversion, a 1,187‐bp amplicon (based on the assay reported in Maglic et al, ) was optimized for use with our routine diagnostic Sanger sequencing workflow (as previously described). Each PCR comprised 0.5 μl of genomic DNA (~50 ng/μl), 14.1 μl of Megamix (Microzone Ltd, Haywards Heath, UK), 0.2 μl of 10 pmol/μl forward primer (dCAGGAAACAGCTATGACCTGACTACGGCCTTGAACTCA) and 0.2 μl of 10 pmol/μl reverse primer (dTGTAAAACGACGGCCAGTGTAACACTGTAGATGCTTTTAG).…”

“…Summary sequencing metrics are recorded inTable S2. Variant calling was performed using NanoPolish (v.0.11.0; https://nanopolish.readthedocs.io) to generate a VCF file, used confirm and genotype the TMEM231 intron 3/exon 4 gene conversion, a 1,187-bp amplicon (based on the assay reported inMaglic et al, 2016) was optimized for use with our routine diagnostic Sanger sequencing workflow (as previously described). Each PCR comprised 0.5 μl of genomic DNA (~50 ng/μl), 14.1 μl of Megamix (Microzone Ltd, Haywards Heath, UK), 0.2 μl of 10 pmol/μl forward…”

Genes and molecular pathways underpinning ciliopathies

“…For example, presumed nonsense mutations in CC2D2A may cause Meckel syndrome (MKS) (MKS6 subtype), whereas missense mutations in the same gene lead to Joubert syndrome (JBTS) (JBTS9 subtype) 188 , suggesting that MKS and JBTS are caused by an allelic series that affects the same essential ciliary function. Similarly, different alleles of TMEM231 are associated with MKS, orofaciodigital syndrome (OFD) and JBTS, even within one family 101,111,189 .…”