Refining genotype–phenotype correlation in Alström syndrome through study of primary human fibroblasts

Abstract

BackgroundAlström syndrome (AS), featuring retinal dystrophy, neuronal deafness, cardiomyopathy, metabolic syndrome, and diffuse fibrosis, is caused by biallelic mutations in the centrosomal protein ALMS1. Genotype–phenotype correlation has been suggested without assessment of ALMS1 expression.Methods ALMS1 expression (real‐time PCR and immunocytochemistry) and cilia formation (immunocytochemistry) were assessed in fibroblasts from deeply phenotyped volunteers diagnosed with AS recruited from a dedicated AS Service. Exome sequencing was used in two participants without convincing biallelic ALMS1 mutations, and BBS2 (Bardet–Biedl syndrome 2) protein expression was assessed in one patient with biallelic BBS2 mutations. Hedgehog‐induced GLI1 expression and PDGFA signaling was assessed using quantitative real‐time PCR, immunoblotting, or immunostaining of fixed cells after stimulation.ResultsIn 16 of the patient cell lines examined, ALMS1 protein was undetectable (14 with biallelic loss‐of‐function (LoF) mutations), and in two, ALMS1 staining was equivocal (one with biallelic LoF mutations). In five lines, ALMS1 expression was normal using at least one fixation method (one with biallelic LoF mutations). These differences were not accounted for by major differences in ALMS1 mRNA expression. Exome sequencing of two participants with normal ALMS1 expression identified biallelic LoF BBS2 mutations in one. No second, known ciliopathy mutation was found in the other patient, who had one LoF ALMS1 mutation. Phenotypes were milder or atypical in participants with preserved ALMS1 immunostaining, even when two with likely alternative genetic diagnoses were excluded. All cells studied developed normal cilia, ALMS1 and BBS2 mutant cells showed normal Hedgehog‐induced upregulation of GLI1 expression, and PDGFA signaling was normal in ALMS1‐deficient cells.ConclusionMilder or atypical presentations of AS should prompt genetic evaluation for alternative, clinically overlapping ciliopathies. A subgroup of patients with bona fide ALMS1 defects have milder phenotypes due to residual ALMS1 expression, which may be more important than mutation site.

ALMS1 and Alström syndrome: a recessive form of metabolic, neurosensory and cardiac deficits

“…The formation of primary cilia seems to be unaffected in AS patient fibroblasts in vitro [19, 76, 142]. Hh and PDGFA signalling, both of which act through the cilium, also appear to be normal [142].…”

“…Hh and PDGFA signalling, both of which act through the cilium, also appear to be normal [142]. In stark contrast, transient RNAi-mediated depletion of ALMS1 has been reported to cause severe defects in ciliary structure.…”

“…It is notable that Alms1 immunostaining was not detected in Alms1 foz/foz neurons [58], nor in fibroblasts from 14 out of 16 AS patients with biallelic nonsense/frameshift mutations [142], implying the absence of residual protein function. Nevertheless, expression of ALMS1 isoforms lacking the antibody epitopes used for detection cannot be excluded in these cases.…”

“…The formation of primary cilia seems to be unaffected in AS patient fibroblasts in vitro [ 19 , 76 , 142 ]. Hh and PDGFA signalling, both of which act through the cilium, also appear to be normal [ 142 ]. In stark contrast, transient RNAi-mediated depletion of ALMS1 has been reported to cause severe defects in ciliary structure.…”

Isoform-specific aPKC renders primary cilia dispensable for Hedgehog signaling and basal cell carcinoma growth

“…As expected, Kif3a KD resulted in loss of primary cilia, a reduction in axoneme length, and suppressed HH signaling compared to a scramble shRNA control ( Figures 1C-F, Supplementary Figure 1A). Alms1 KD phenocopied Kif3a KD, despite normal primary ciliogenesis in Alms1 -/mice that present similar features as patients with Alström syndrome (Collin et al 2005) and in primary human fibroblasts cultured from Alström syndrome patients (Chen et al 2017). Despite not previously linked to primary cilia or HH signaling, KD of Usher Type 1 genes Cdh23 and Pcdh15 or Usher Type 2 genes Gpr98 and Ush2a also resulted in ciliary loss, reduced axoneme length, and diminished HH signaling compared to scrambled shRNA control ( Figures 1C-F, Supplementary Figure 1A).…”