In the present short report, Mahmood et al, has recruited a family segregating Limb Girdle Muscular Dystrophy (LGMD). They performed whole exome sequencing (WES) of a proband and his parents to identify the disease-causing variant. Analysis of WES revealed a novel homozygous variant in POPDC1. The gene has been previously associated with LGMD type 25. In addition to WES, the authors performed in silico analysis of previously reported and the identified missense variants to check its effects on protein structure and function.
Overall, the report is well structured and written. I have several comments that might be helpful in the improvement of manuscript.
Add full name Limb Girdle Muscular Dystrophy instead of LGMD in title.
In title, the authors write “biallelic variant”. They have exome data of parents and proband, therefore, it would be interesting to calculate runs of homozygosity (ROH) and check whether the variant is homozygous. If the variant if present in ROH, then “biallelic” should be replaced with “homozygous”.
Abstract; Page 3; line 24: Add “type 25” between limb-girdle muscular dystrophy and (LGMDR25). In addition, check if it is either LGMD or LGMDR?
The authors have used abbreviations at some points without writing full name at its first appearance, e.g.,
Page 3, line 31: MD…? etc.
Page 3, Line 33: Add NM_ and NP_ numbers to the identified variant in abstract.
Page 5, line 34: Citation [9, 10] to ANNOVAR is not correct.
Page 5, line 42-48: “Variants having minor allele frequency more than 0.01 in any of human genome databases such as dbSNP (https://www.ncbi.nlm.nih.gov/snp/), 1000 Genomes (https://www.internationalgenome.org/) and NHBLI 6500 (https://evs.gs.washington.edu/EVS/) exomes were selected.” Please check, variants having minor allele frequency less than 0.01 should be selected for further analysis.
Page 5; Line 53: (ii) “thought to be responsible for LGMD”. It is not clear. How did the authors think of a gene to be responsible for LGMD, I mean based on what?
Page 6, Line 3: “exhibited a plausible pattern of inheritance in the pedigree [2].” The present pedigree could be autosomal recessive, X-linked and de novo or even a two hit model. Therefore, neither the pedigree depicts a clear inheritance pattern nor the citation [2] reports anything that can support the statement.
Page 6, Line 9: Sanger Sequencing: The authors have sequenced proband and his parents using whole exome sequencing. None of the normal siblings from the pedigree has been Sanger sequencing. Therefore, it is not clear why the authors state that Sanger sequencing was performed to assess the segregation of the variant c.401C>T. Maybe, the Sanger sequencing was performed to validate the WES results.
Page 6, line 10; c. 401C>T or c.401C>T?
Page 6, line 41: Remove full stop after preparation
Page 7 line 56: “The mother felt weak muscles, hands grip, and walking.” The statement is not clear. When did the mother feel weak muscles………….
Page 8; line 8: Add full creatine kinase before CK. Also, add reference value for CK.
Page 8, line 10: “An overview of clinical description of present study along with literature is given in Table1.” Add space between table and 1.
Page 8, line 46: Brain Magnetic resonance imaging (MRI) or Brain Magnetic Resonance Imaging (MRI)?
Page 9; line 13: Section: Homozygous missense variant in POPDC1 gene
Here, the authors should add total number of homozygous and heterozygous variants left after applying filter of MAF<0.01. In addition, were there any heterozygous variants in other genes related to LGMD that can act as modifiers?
Page 9, line 17-22: The authors state “We focused on pathogenic, likely pathogenic, nonsynonymous (missense, nonsense) along with splice sites, frameshift and variants of unknown significance (VUS) variants.” It is not clear, how did they classify the variants into pathogenic, likely pathogenic and VUS.
Page 9, line 25-27: “Using our prioritization criteria and already reported genes associated with LGMD/MD (Table S1), we selected 30 genes (already reported) for validation (see Table S1).”
Why the authors restricted selection criteria to previously LGMD-associated genes? Were there variants in these genes in exomes of the present individuals?
Page 9, line 44: genomeAD? Please correct.
Page 9, Line 46: Add “in POPDC1” after amino acid Proline at position 134.
Page 10, line 23-25: Please correct headings.
Page 10, line 49: 40 ns and 60ns. Please correct.
Page 10, line 54: Please add full stop after system instead of comma.
Page 12; line 10: “Did not revealed”. Please correct the sentence
Page 12: Line 10: Patients or patient?
Page 12: Line 12: Please add full name for CNS
Page 12; line 45: Please remove brackets from (p.Pro134Leu).
Page 13: Line 3: Add space between POPDC1 and Similarly.
Page 16: Figure 1A: Add description for arrow and asterisk signs.
Page 16: Figure 1B, Add arrows to figure 1B to show abnormality in brain.
Page 16: Figure E-G: Poor quality of chromatograms.
Page 16: Legends to E-H is not correct, Figure 1I is either missing or the legends are for Figure 1H instead of 1I.
Page 17: Figure 2: Zoom-In?
Page 19; Table 1: The table does not report all the variants identified in POPDC1 till date, e.g. variants reported by Schindler et al. (2016) Gangfuss et al. (2022).
Page 20; Table 2: Add ACMG interpretation of the identified variant.