Peer review of: Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is a thoroughly studied, well described and well written paper reporting 20 years of study 
leading to the identification of hypomorphic hemizygous variants in FAM50A as disease-causing in 
Armfield syndrome, an X-linked recessive syndromic form of ID. The authors also decipher the role 
of Fam50a in splicing regulation via transcriptomic analysis of wt and ko zebrafish larvae. It adds 
an animal model to tackle the fascinating question of the specificity of phenotypes resulting from 
spliceosomopathies. 
I have only minor comments : 
The involvement of FAM50A in the spliceosome complex should be stated in the introduction 
section. 
“recessive variant” should be replaced by hemizygous variant 
Selection criteria for likely causal changes are not specified. 
line 120 : “to validate the specificity of this finding” do you mean that there are no other candidate 
variant? 
line 120 to 124: 2 sentences giving the same information. delete one 
Is there data regarding the inactivation of FAM50A? Is there an inactivation bias in female 
carriers? 
Isn’it the right time to change the gene Name? 

Reviewer #2 (Remarks to the Author): 

The manuscript reports the identification of rare variants in Fam50A, likely causing Armfield XLID 
syndrome in human. Functional evidence for the gene being involved in this severe syndrome is 
obtained using a zebrafish genetic KO. The genetic datasets presented are supporting the 
involvement of the Fam50A in the patient pathologies. However, I would recommend 
strengthening the functional evidence from the zebrafish model before considering publication 
further. The conclusion that mutations in FAM50A induce spliceosomopathy is a vast over-
interpretation of the current datasets presented in this manuscript. Specific major comments are 
listed below. 

Using the zebrafish mutant to assess the effect of human mutations: 
- The human mutations found in patients are predicted to be hypomorphs. Using an animal model 
losing protein function completely is therefore only indirectly informative. The KO has to be used 
more directly to assess the phenotype imposed by the patient mutation, by rescuing the null 
embryos with the human variants. The authors can rescue the mutant be either wildtype or 
mutant Fam50A mRNA injection (or make a series of UAS:Fam50A transgenics – this latter 
approach being too long-winded for a rapid publication). Rescue by RNA should be possible as the 
defects start before day 2 (cell death is detectable at 48hpf already, as shown in Fig. 2D). 
Moreover, they could also use DNA vectors expressing tagged FAM50A (comparing wt and 
variants), which will be expressed mosaically and monitor the behaviour of progenitors and 
neurons in the null. This is a crucial set of experiments needed to understand the impact of the 
patient isoforms on the CNS. 
- The MO experiments described in Fig. 3 are too indirect to properly address the nature of the 
human mutations. The experiments above need to be done instead. 

When is the defect initiated in the zebrafish zygotic nulls: 
- As the zebrafish Fam50A is expressed maternally, it is important to know when the protein 
becomes undetectable in the zygotic null mutants. 
- As cell death is dramatically increased in cell populations known to be Wnt-secreting (Fig. 2D) as 
early as 48hpf, the authors need to follow what is happening to the brain of these mutants 

between 24 and 48hpf, looking at HuC and her4 transcripts (the Tg(huc:egfp) line accumulates 
GFP over time, masking subtle differences). In Fig. 2C, her4 looks weaker in the tectum already at 
48hpf. 

How specific and direct is the effect on RNAs involved in splicing?: 
- Apoptosis has been shown to up-regulate of spliceosome proteins and more generally RNA 
processing proteins, generating distinct isoforms of apoptotic regulators through alternative 
splicing. The localisation of these RNAs (Fig.5) to the areas of cell death (Fig. 2D) supports this 
idea. The authors need to test by PCR whether blocking apoptosis in the mutant reduce the level of 
upregulation of splicing proteins and the % increase of alternative 3’ UTR events. 

Reviewer #3 (Remarks to the Author): 

Armfield XLID syndrome was previously linked to an 8 Mb region on Xq28. In this study, Lee et al 
describe FAM50A rare missense variants (D255G, W206G, E254G, R273W, D255N) in 9 affecteds 
from multiple families with clinical presentations related to Armfield XLID. To clarify the effects of 
these variants in a vertebrate model, they generate fam50a KO zebrafish models that reproduce 
some neurodevelopmental and craniofacial patterning defects observed in this XLID syndrome. 
Complementation assays revealed 5 non-synonymous hypomorphic changes and RNAseq indicated 
changes in 12% of the transcriptome, with a majority of 3’ss shifts, resulting in loss of RNA 
transcripts important for brain development. Overall, this is an interesting analysis of additional 
XLID cases, although the emphasis of this study is on characterization of fam50a KO zebrafish that 
reproduce some features of Armfield XLID. Additionally, their only experimental readout is 
abnormal splicing, and the evidence for a direct role of FAM50A in splicing regulation and/or 
spliceosome function is incomplete. 

Major: 
1. The progression from discovering FAM50A missense variants that show clinical phenotypes 
similar to Armfield XLID variants to studying these variants in zebrafish is a reasonable approach. 
However, the zebrafish KO phenotype is the most striking result so I examined the FAM50A 
structure and noticed a 5’ UTR GCC microsatellite just 5’ to the translation initiation codon 
suggesting the possibility of a pathomechanism similar to FRAXA. Have the authors performed 
Southern botting to determine if there is a GCC expansion mutation in Armfield XLID? 

2. Results, p7/8 and Fig. S3. Since FAM50A is ubiquitously expressed in fetal and adult tissues, do 
Armfield XLID patients show a change in the expression level of this gene? 

3. Results, p10, ln257. The assertion that the early neurogenesis defects observed in fam50sa KOs, 
with lethality at ~6dpf in homozygotes, are directly relevant to Armfield XLID is an overstatement. 

4. Results, p14, ln353 and Figs. 5 and S13. First, the selection criteria for analyzing these genes 
was unclear – were these the top 25 genes mis-regulated in the KO? Second, it is interesting that 
alternative 3’ss changes are the predominant abnormal splicing event, but the authors fail to 
provide convincing evidence for a a direct role of FAM50A in splicing and spliceosomal function and 
explain why changes in 3’ss selection are primarily affected. 

Minor: 
5. Abstract. Alterations in splicing leading to alterations in splicing patterns are generally referred 
to as spliceopathy not spliceosomopathy. 

6. Fig. S4A. Using the anti-FAM50A antibody in 3T3 cells, FAM50A protein appears equivalently 
distributed throughout the nucleus whereas the V5-tagged version in Cos7 cells is concentrated at 

the nuclear periphery/NE. The authors should comment on this discrepancy. 

7. Fig. S5B. Provide a rationale for CRISPR targeting exons 6,7 to generate KO alleles?