Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

Abstract

Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in COL4A3, COL4A4, and COL4A5 genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting COL4A5 exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel COL4A5 hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in COL4A3 exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.

X-Linked Alport Syndrome in Women: Genotype and Clinical Course in 24 Cases

“…However, it is well-known that the same mutation, whether moderate or severe, can give phenotypic variability even within the same sex. Considering our population, Table 3 shows that the age of appearance of ESKD in the male relatives of our patients is not statistically different between the two groups ( p = 0.711), but the early appearance of ESKD (<35 years of age) confirms that the male phenotype is usually more serious than the female one sharing the same mutation, in which the inactivation of X must always be taken into account ( 13 ).…”

“…She developed proteinuria at 13 years of age. Using a minigene-based approach in HEK293 cells, we previously demonstrated that this variant abolishes exon29 branch site ( 13 ). The variant was also present in her mother (Patient n°2), who showed only microscopic hematuria.…”

“…The variant was also present in her mother (Patient n°2), who showed only microscopic hematuria. This difference in the phenotype between mother and daughter correlates with the levels of inactivation of the X-chromosome carrying the wild type allele, as assessed by a methylation-sensitive restriction-enzyme assay ( 13 ). Indeed, the wild-type c.2245-40A allele showed a very high level of inactivation (91%) in the proteinuric daughter (Patient n°14), whereas the non-proteinuric mother (Patient n°2) had a balanced inactivation of both alleles (45% vs. 55%).…”

“…Skewing of XCI in two female carriers of the COL4A5 c.2245-40A>G mutation was investigated by analyzing a polymorphic trinucleotide repeat in the androgen receptor gene, for which the tested females were heterozygous, by means of a methylation-sensitive restriction enzyme assay, as previously described ( 13 ).…”

Novel inactivating mutations in the FSH receptor cause premature ovarian insufficiency with resistant ovary syndrome

“…Efficient cleavage by the signal peptidase requires small and aliphatic residues (in particular Ala, Gly, Ser and Val) at the 1-and 3-positions of the cleavage site (Choo and Ranganathan, 2008;Lindert et al, 2017). Therefore, the Asp15 substitution in FSHR, at the 3-position of the cleavage site, might lead to subcellular mislocalization of FSHR (Bornemann et al, 2008;Chiereghin et al, 2017;Halic et al, 2006;Holtkamp et al, 2012;Rutz et al, 2015).…”