during embryogenesis , the small intestine experiences major growth expanding several times the total length of the whole body . this is a complex process that starts around the fifth week of human development , when the midgut forms a simple linear tube that runs down the midline of the embryo , and is completed by the twentieth week of development1 . several pathways have been reported to play a role in intestinal elongation2 , but to date the molecular mechanism(s ) responsible for this process remains unclear . in congenital short bowel syndrome ( csbs ) intestinal elongation is impaired , and patients are born with a substantially shortened small intestine ( approximately 50 cm in length instead of 250 cm in a normal counterpart)3 . it is a rare disorder for which no cure is available . total parenteral nutrition is required for long - term survival , but despite considerable efforts to improve treatment , most patients die of starvation or sepsis within the first few days of life4 . csbs was first described by hamilton et al . in 1969 , and it has always been considered to have a genetic cause3 . however , it was only recently that two genes were identified as the cause of csbs : clmp and flna5,6 . clmp encodes for the coxsackie - and adenovirus receptor - like membrane protein , an adhesion molecule that co - localizes with tight junction proteins , but whose function is still unknown7 . flna encodes for filamin - a , an actin binding protein known to regulate cell shape and to control cell signalling and migration8 . pathogenic variants present in clmp and flna have been linked to the recessive and x - linked forms of csbs , respectively . here , we report the genetic screening of three csbs patients derived from two unrelated families , and describe the identification of three novel variants in clmp . in this study , three female patients diagnosed with csbs were investigated ( table 1 ) . two of these patients were 
 siblings of consanguineous descent ( p1 and p2 ) . the third patient ( p3 ) has been 
 previously described , when a de novo translocation was found 
 between chromosome 2 and 11 ( 46,xx , t(2,11)(q32.2,p12))9 . written informed consent was given by the two families reported , and ethical approval was obtained from the erasmus medical center ethical committee ( medisch ethische toetsings commissie - metc 2009/364 , abr nr : nl31708.042.10 ) . exons 1 - 7 of clmp ( enst00000448775 ) were amplified using 30 ng of genomic dna , as described before5 . ge healthcare , eindhoven , the netherlands ) , and sanger sequencing was performed with dye labelled primers ( forward and reverse ; big dye terminator v3.1 sequencing kit , applied biosystems , bleiswijk , the netherlands ) on an abi 3130xl genetic analyzer . sanger reads were analysed using seqscape software and compared to the reference clmp genomic sequence ( ensg00000166250 ) . genetic data was submitted to clinvar ( http://www.ncbi.nlm.nih.gov/clinvar/ ) and the following accession numbers were obtained : scv000264793 [ p.(r170 * ) ] and scv000264792 [ p.(c137y ) and ( c.29 - 2a > g ) ] . five hundred thousand cells were seeded in a 6-well plate , and transiently transfected with pcdna - ha - clmp wt or pcdna - ha - clmp c137y expressing vectors , using genejuice ( millipore , amsterdam , the netherlands ) as transfection reagent . images were taken in a leica ( aobs ) microscope , and analysed with the leica las af lite software . in this study , three female patients diagnosed with csbs were investigated ( table 1 ) . two of these patients were 
 siblings of consanguineous descent ( p1 and p2 ) . the third patient ( p3 ) has been 
 previously described , when a de novo translocation was found 
 between chromosome 2 and 11 ( 46,xx , t(2,11)(q32.2,p12))9 . written informed consent was given by the two families reported , and ethical approval was obtained from the erasmus medical center ethical committee ( medisch ethische toetsings commissie - metc 2009/364 , abr nr : nl31708.042.10 ) . exons 1 - 7 of clmp ( enst00000448775 ) were amplified using 30 ng of genomic dna , as described before5 . ge healthcare , eindhoven , the netherlands ) , and sanger sequencing was performed with dye labelled primers ( forward and reverse ; big dye terminator v3.1 sequencing kit , applied biosystems , bleiswijk , the netherlands ) on an abi 3130xl genetic analyzer . sanger reads were analysed using seqscape software and compared to the reference clmp genomic sequence ( ensg00000166250 ) . genetic data was submitted to clinvar ( http://www.ncbi.nlm.nih.gov/clinvar/ ) and the following accession numbers were obtained : scv000264793 [ p.(r170 * ) ] and scv000264792 [ p.(c137y ) and ( c.29 - 2a > g ) ] . chinese hamster cells ( cho - k1 ) were cultured as described before5 . five hundred thousand cells were seeded in a 6-well plate , and transiently transfected with pcdna - ha - clmp wt or pcdna - ha - clmp c137y expressing vectors , using genejuice ( millipore , amsterdam , the netherlands ) as transfection reagent . images were taken in a leica ( aobs ) microscope , and analysed with the leica las af lite software . as the three patients included in this study are females , an x - linked pattern of inheritance involving flna was considered unlikely , and only clmp was screened . in all patients we identified previously unreported variants in clmp . none of these variants is listed in any of the available human genome variant databases . patients p1 and p2 possess a homozygous nonsense variant in exon 4 [ c.508c > t ; p.(r170 * ) ] , leading to the appearance of an early stop codon ( fig.1a ) . this nonsense variant was inherited from the parents , who were found to be heterozygous . although we suspect that both variants were inherited from the parents , we were unable to investigate segregation in the family due to unavailability of parental dna . in this patient a missense variant located in exon 4 was identified leading to an amino acid substitution [ c.410g > a ; p.(c137y ) ] , together with a possible splice site variant located two base pairs upstream of exon two ( c.29 - 2a > g ) ( fig.1a ) . prediction programs used to infer pathogenicity ( polyphen-2 , mutationtaster , sift , human splicing finder and alamut visual ) showed that both variants are likely to disturb protein function . the c137y variant is located in a highly conserved region present in all mammals ( fig.1b ) , and in vitro assays showed that it affects the cellular localization of clmp to the tight junctions ( fig.1c ) . the c.29 - 2a > g variant is predicted to disrupt normal splicing of exon 2 of clmp ( fig.1d ) . considering that both variants likely affect clmp function , we believe that the de novo translocation previously identified in this patient9 is an independent event unrelated to csbs . all the variants reported were loss of function , leading to the absence of clmp , or to the expression of a mutant protein with a disrupted function ( fig.1e ) . in this report , we expand the mutational spectrum of clmp variants involved in csbs , consolidating the involvement of this gene in the development of the recessive form of this disease . we also show that it might be worthwhile to screen previously described patients for whom an x - linked pattern of inheritance has been disregarded , for the presence of variants in clmp , as this might shed some light on the genetic basis of the disease in these cases . to date , the role of clmp in intestinal elongation is still largely unknown , making it difficult to determine the molecular mechanisms underlying the development of csbs . we believe that the results here described reinforce the need to further study the role of clmp in intestinal elongation , as this might contribute for the development of new therapeutic strategies for csbs patients .
congenital short bowel syndrome ( csbs ) is an intestinal pediatric disorder , where patients are born with a dramatic shortened small intestine . 
 pathogenic variants in clmp were recently identified to cause an autosomal recessive form of the disease . however , due to the rare nature of csbs , only a small number of patients have been reported to date with variants in this gene . in this report 
 , we describe novel inherited variants in clmp in three csbs patients derived from two unrelated families , confirming clmp as the major gene involved in the development of the recessive form of csbs .