Disruption of SMIM1 causes the Vel− blood type

Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti-Vel regularly causes severe haemolytic transfusion reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel− cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel− individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel− blood type, likely having a common origin, facilitated the development of two highly specific DNA-based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60-year-old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel− patients.

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I look forward to receiving your revised manuscript. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): This is an important contribution to our knowledge of blood group antigens and one of the last antigens to be characterized. The Vel antigen is rare but because it can cause strong hemolytic reactions understanding the molecular genetics and biochemistry is important for transfusion safety and development of typing reagents and methods.
Referee #1 (General Remarks): This manuscript describes the molecular genetic basis of the Vel blood group antigen -a rare and important antigen that can cause severe hemolytic reactions, which has been a puzzle for over 50 years. The authors use a potent anti-Vel antiserum that Western blots to isolate the 32/18 kd protein and partially sequence it by de novo mass spec sequencing of trypsin in-gel digests. The sequences identified matched a predicted small protein with a single hydrophobic sequence in the C-terminal region, and sequencing of the gene (SM1M1) in Vel positive and Vel negative individuals demonstrated a 17 nt deletion in Vel negatives, confirming the identity of this protein as representing the Vel antigen. This was further substantiated by expression of the gene in K562 cells. The experimental design and results are clear and conclusive and the manuscript is well written. The molecular genetic basis of the Vel blood group antigen is important for the transfusion field and will have wider general interest.
The first sentence in the abstract claims that the report includes the biochemical basis of the Vel antigen (not in the title!), but this may be argued as the antigenic epitope is not clarified per se. This reviewer would suggest that a discussion of the potential epitope is included. This could include a discussion of the unusual predicted topology of the protein without N-terminal signal sequence (does the C-terminal hydrophobic domain serve as non-cleavable signal peptide?) and a correlation of predicted mass and the estimated mass. It appears that the protein is only 78 amino acids (include The study required a high level of technical expertise in order to isolate the protein but the metods required. Identification of Vel protein and gene, and the molecular basis of the Vel-negative phenotype. This information is valuable for confirming Vel-negative phenotype and for screeing for Velnegative donors. As extended blood group phenotyping of blood donors by molecular methods is starting to be introduced widely, this will provide a useful addition. I must declare that my expertise lies in blood group genetics and I am not qualified to comment on the biochemical analyses employed.

Referee #2 (General Remarks):
This is a well-written and interesting manuscript describing the identification of the gene encoding the Vel blood group antigen and the molecular background of the rare Vel-negative phenotype. I only have a few minor comments.
Title VEL is not a novel blood group system. Although the findings reported in this paper are novel, Vel is a blood group antigen that has been known for decades. A VEL system may be created by the International Society of Blood Transfusion as a result of these findings, but this is not mentioned in the paper.

Introduction
Page 3, bottom line. I am not sure what is meant by 'notorious obstinance' (and I don't think that obstinance is a word). This should be changed. The term 'high-titer, low avidity' is outdated and unhelpful. In addition, even when the term was used, it was not usually applied to anti-Vel. The main reason why many examples of anti-Vel are difficult to detect is that they are IgM and only reactive in an antiglobulin test when a source of complement is present.

Results
Page 8, end of first paragraph. This is misleading. I know of no evidence that Vel expression is lost with age. The comparison with JMH is irrelevant. Page 8, second paragraph. The authors claim that the 17 nt deletion is the predominant cause of Velnegative and suggest that this demonstrates a founder effect. However, they do not mention the ethnicity of the Vel-negative individuals tested. Vel-negative phenotype has been found in ethnic groups other than Caucasians, for example Thais and native Americans, and they may have other mutations. If the authors are unable to test Vel-negatives from other ethnic groups, they should state the ethnicity of those tested. If they do not know and are unable to find out, they should discuss the issue. The first sentence in the abstract claims that the report includes the biochemical basis of the Vel antigen (not in the title!), but this may be argued as the antigenic epitope is not clarified per se. This reviewer would suggest that a discussion of the potential epitope is included. This could include a discussion of the unusual predicted topology of the protein without N-terminal signal sequence

(does the C-terminal hydrophobic domain serve as non-cleavable signal peptide?) and a correlation of predicted mass and the estimated mass. It appears that the protein is only 78 amino acids (include Fig of sequence and predicted topology?), which is difficult to envision running at 18/32 kd as mono/dimer?
As suggested by this reviewer, we have included in the Discussion section the hereafter paragraph to discuss the potential epitopes of anti-Vel in the SMIM1 protein, the absence of a signal peptide for this single-pass membrane protein, as well as the difference between its theoretical molecular weight and its observed molecular weight in SDS-PAGE.
'While the exact epitope recognized by anti-Vel remains to be defined, the small size, as well as the predicted structure of SMIM1 with a single transmembrane domain, limits the number of potential epitopes. Toward this goal, it would be important to determine whether SMIM1 is a type I or II membrane protein, i.e., whether its N-terminus is extra-or intracellular. Of note, most type I membrane proteins have a N-terminal cleavable signal peptide in contrast with type II membrane proteins, whose transmembrane domain functions as a (non-cleavable) signal peptide and as a membrane anchor. Our mass spectrometry identification of extreme N-terminal SMIM1 amino acids is consistent with SMIM1 being a type II membrane without a N-terminal cleavable signal peptide. One may also note that SMIM1 does not migrate at its theoretical molecular weight (8.7 kDa) in SDS-PAGE even under reducing conditions. This may reflect the presence of post-translation modifications including glycosylations or phosphorylations. Nevertheless, one should not neglect that membrane proteins often deviate from their theoretical molecular weight in SDS-PAGE as hydrophobic transmembrane domains bind less SDS (Rath et al, 2009).'

Referee #2 (Comments on Novelty/Model System):
The study required a high level of technical expertise in order to isolate the protein but the methods required.

Identification of Vel protein and gene, and the molecular basis of the Vel-negative phenotype. This information is valuable for confirming Vel-negative phenotype and for screening for Velnegative donors.
As extended blood group phenotyping of blood donors by molecular methods is starting to be introduced widely, this will provide a useful addition. I must declare that my expertise lies in blood group genetics and I am not qualified to comment on the biochemical analyses employed.

Referee #2 (General Remarks):
This is a well-written and interesting manuscript describing the identification of the gene encoding the Vel blood group antigen and the molecular background of the rare Vel-negative phenotype.

I only have a few minor comments.
Title VEL is not a novel blood group system. Although the findings reported in this paper are novel, Vel is a blood group antigen that has been known for decades. A VEL system may be created by the International Society of Blood Transfusion as a result of these findings, but this is not mentioned in the paper.
We totally agree that the Vel blood group antigen has been known for decades, as clearly stated in our manuscript ('The existence of the Vel antigen was recognized in 1952 by Sussman and Miller who found an alloantibody with a novel specificity in the serum of Mrs. "Vel" who suffered an acute HTR (Sussman & Miller, 1952).'). Our manuscript reports the identity of the protein carrying the Vel antigen, the identity of the gene encoding the Vel antigen, and the identity of a predominant mutation responsible for the Vel− blood type, hence specifying a novel blood group system according to the International Society of Blood Transfusion (ISBT). Nevertheless, this reviewer is right when saying that VEL is not (yet) a novel blood group system until the ISBT officially creates

Introduction Page 3, bottom line. I am not sure what is meant by 'notorious obstinance' (and I don't think that obstinance is a word).
This should be changed.
'Notorious obstinance' has been replaced by 'notorious reluctance' to elegantly express that most immunohematologists think that anti-Vel is a very difficult antibody to work with in vitro.
The term 'high-titer, low-avidity' is outdated and unhelpful. In addition, even when the term was used, it was not usually applied to anti-Vel.
We thank the reviewer for this comment but we respectfully disagree. The term 'high-titer, low affinity' (HTLA) is still used extensively, not only in the French National Reference Center for Blood Groups but also in many other European reference laboratories, even though some immunohematologists think that this term is outdated. As a matter of fact, the HTLA characteristics of anti-Vel are mentioned in the We thank the reviewer for this technical comment.
Page 8, end of first paragraph. This is misleading. I know of no evidence that Vel expression is lost with age. The comparison with JMH is irrelevant.
We thank the reviewer for sharing professional experience. Our comment was made to propose a potential explanation by mentioning that 'The advanced age of this subject may explain why her RBCs did not express the Vel antigen anymore, as is often observed for the JMH blood group antigen in elderly people'. We made the comparison with JMH on purpose because this blood group antigen is the paradigm of expression loss with age. We leave the decision to the editor to keep or remove this sentence. We thank the reviewer for reminding us about the discovery in the 60s' of four Thais and two Chilcotins with the Vel− phenotype. As suggested by this reviewer, we have included in the Discussion section the hereafter paragraph to discuss our findings as well as these historical findings from an ethnological point of view.
'Even though almost all the Vel− persons reported so far were of European descent including our cohort of 70 Vel− subjects, a few cases among other ethnicities have been reported. Four Thai persons were identified as Vel− in 1967 during an extended phenotyping of 328 blood donors from Bangkok (Chandanayingyong et al, 1967). It would be interesting to determine whether or not the Vel− phenotype in persons of Thai descent results from the same SMIM1 disruption. Two out of 160 Chilcotin First Nations people (British Columbia, Canada) were also reported to be Vel− but this result should be interpreted with caution as the authors of this ethnologically oriented study did not mention their source of anti-Vel (Alfred et al, 1970). As a matter of fact, a similar study of 133 Penobscot Native Americans (Maine, USA) with a potent anti-Vel revealed no Vel− subjects "though two siblings were such weak positives that they were at first thought to be negative" (Allen & Corcoran, 1960).'