Autosomal recessive congenital short bowel syndrome is caused by mutations in CLMP. No mutations were found in the affected males of a family with presumed X-linked congenital short bowel syndrome or in an isolated male patient. Our aim was to identify the disease-causing mutation in these patients.
We performed mutation analysis of the second exon of FLNA in the two surviving affected males of the presumed X-linked family and in the isolated patient.
We identified a novel 2-base-pair deletion in the second exon of FLNA in all these male patients. The deletion is located between two nearby methionines at the N-terminus of filamin A. Previous studies showed that translation of FLNA occurs from both methionines, resulting in two isoforms of the protein. We hypothesized that the longer isoform is no longer translated due to the mutation and that this mutation is therefore not lethal for males in utero.
Our findings emphasize that congenital short bowel syndrome can be the presenting symptom in male patients with mutations in FLNA.
Genet Med 2013:15(4):310–313
Congenital short bowel syndrome (CSBS) is characterized by a shortened small intestine and intestinal malrotation. Although the normal length of the small intestine at birth is ~275 cm,1 patients with CSBS have a markedly shorter small intestinal length of ~50 cm, on average. CSBS has a high mortality rate within the first few months after birth, although some long-term survivors of CSBS have been reported.2,3,4,5
CSBS is an inherited disorder. The identification of homozygous and compound heterozygous mutations in Coxsackie- and adenovirus receptor-like membrane protein (CLMP) in CSBS patients confirmed an autosomal recessive pattern of inheritance in most affected families.6 However, no CLMP mutations were identified in one Italian family7,8 or in an isolated German-American male ( Table 1 ) who presented with congenital short bowel syndrome.5 In the affected family, only males developed the disease, consistent with an X-linked pattern of inheritance ( Figure 1 ).8
In the literature, a family with X-linked chronic idiopathic intestinal pseudo-obstruction (CIIP) has been described.9,10 The patients in this family have very similar features to those seen in patients with CSBS with mutations in CLMP, including a shortened small intestine. A 2-base-pair (bp) deletion in the second exon of filamin A (FLNA) (c.65-66delAC) was identified in this X-linked CIIP family. This deletion is located between two nearby methionines at the N-terminus of FLNA. It was shown that translation of FLNA can occur from either methionine in vitro and that the deletion affects only the longer form of FLNA.10
We hypothesized that the longer form of FLNA is essential for normal small intestinal development. Therefore, we performed mutation analysis of the second exon of FLNA in the X-linked CSBS family and in the isolated male patient.
Materials and Methods
The presumed X-linked CSBS family was reported by Kern et al.8 As described in their case report, the patients in this family presented with bile-stained vomiting and diarrhea, symptoms typically seen in patients with CSBS. The two patients in the Italian family who survived were included in our study. Patient II-6 was born with a small intestine of a total length of 60 cm. The family pedigree is presented in Figure 1 . The isolated male patient (patient I, family 2) who presented with CSBS was described by Siva et al.,5 who emphasized the extensive nature of a rare arthropathy known as synovial lipomatosis. The length of the small intestine in this patient was 90 inches (228.6 cm) at the age of 15 (one-third of normal length). This patient is a long-term survivor and is >40 years old. The arthropathy was reported, but it did resolve spontaneously.
Our study protocol was approved by the institutional and national ethics review committees at the University Medical Centre Groningen (NL31708.042.10) and at the Washington University School of Medicine. Written informed consent was obtained from all the study participants.
Genomic DNA was isolated from peripheral whole-blood lymphocytes by standard procedures. Mutation analysis of the exonic and flanking intronic regions of the second exon of FLNA (NG_011506.1) was performed using the primers FLNA forward, 5′-IndexTermCGCAACCTCTGCTCCCTGCC-3′, and FLNA reverse, 5′-IndexTermGCGCCACCGACACGTTCTCA-3′. PCR was performed as follows: 35 cycles with 100 ng of genomic DNA at 95 °C for 1 min, at an annealing temperature of 55 °C for 1 min and then at 72°C for 1 min. This was accomplished in two patients of the family and their unaffected relatives, in the isolated male patient (patient I, family 2) and his mother, and in 92 controls of Caucasian ethnicity.
We identified a 2-bp deletion (c.16-17delTC) in the two surviving affected males in the family and also in the isolated male patient ( Figure 1 ). We confirmed co-segregation of the 2-bp deletion in the X-linked family and showed that all the obligate carriers were heterozygous for this deletion. Because the mother of the isolated male patient did not carry the deletion, we concluded that his mutation had occurred de novo. Moreover, this mutation was absent in 92 controls and is not reported in any single-nucleotide polymorphism database or in the exome sequence variant database (http://evs.gs.washington.edu/EVS/).
The c.16-17delTC deletion results in a frameshift and a premature stop codon at amino acid 103. In the predicted protein, only the first six amino acids are retained, which are identical to the wild-type filamin A. They are followed by 97 different amino acids. As the c.16-17delTC mutation is located between the first and second methionine, it likely has a similar effect to the c.65-66delAC mutation (found in CIIP)10 that results in loss of only the long form of FLNA.
Mutations in FLNA are associated with a wide spectrum of disorders, including periventricular nodular heterotopia, otopalatodigital syndromes types 1 and 2, frontometaphyseal dysplasia and Melnick Needles syndrome, and X-linked cardiac valvular dystrophy.11,12,13 Our findings add CSBS to this list as a possible presenting phenotype in male patients with a mutation in FLNA. We therefore emphasize the importance of FLNA in intestinal development. The index patient of the family described by Gargiulo et al.10 developed asymmetrical spastic diplegia, and an abnormal intermediate signal in the peritrigonal white matter was seen on magnetic resonance imaging. We cannot exclude involvement of the central nervous system in our patients because no magnetic resonance imaging brain scans were available. However, they did not have any clinical neurological abnormalities such as seizures or spasticity; not all patients with mutations in FLNA have central nervous system involvement. The mother of the proband described by Kapur et al.14 did have a duplication of the first 28 exons of FLNA, but had a normal cranial magnetic resonance imaging.
In addition to the finding of Gargiulo et al.10 of a 2-bp deletion in FLNA in one male patient with CIIP, Kapur et al.14 reported FLNA mutations in patients with CIIP. They identified a partial duplication of the first 28 exons of FLNA in one family and a nonsense mutation (c.7021C→T; Q2341X) in exon 43 in another patient. However, these patients were diagnosed with multiple congenital anomalies, of which a congenital short bowel was only one feature.14 These findings support our hypothesis that FLNA is important for normal small intestinal development.
As the mutations in the second exon are located between the first two methionines, they probably act as mild mutations, conserving the reading frame encoding the short isoform, associated with a rather mild phenotype. The mutations identified in the patients reported by Kapur et al.14 were much more severe, explaining their more severe phenotype.
Our finding raised the question of whether CSBS patients with a mutation in the second exon of FLNA and CSBS patients with mutations in CLMP have the same phenotype. Clearly, the congenital short bowel is a feature they have in common, but pseudo-obstruction has also been described in CSBS patients with mutations in CLMP. Therefore, the bowel tissue of patients with CSBS has been studied to determine if there is an abnormality of the enteric nervous system underlying the reduced bowel movements. Although the bowel wall in patients with CSBS seems to be macroscopically normal, Tanner et al.15 described abnormal histology of the bowel wall revealed by silver staining, in which there were too many neurons in the ganglia. The neuronal nuclei showed clumped chromatin, which is characteristic of neuroblasts. They observed that the intrinsic argyrophil ganglia were absent or much reduced in number, and argued that these histological findings might cause the motility abnormalities described in their patient as well as in other cases.15 In addition, Schalamon et al.16 observed an abnormal bowel wall with signs of neuronal intestinal dysplasia in a patient from a consanguineous Turkish family, in whom a truncating homozygous mutation in CLMP was detected.6 In other cases, no abnormalities of the nerve plexus were observed on routine histology or by acetylcholinesterase staining.17,18 However, histology specimens were not available for all cases.
To conclude, the clinical features of CSBS patients with FLNA mutations conserving the short isoform and CSBS patients with mutations in CLMP are very similar. Both have a congenital shortened small intestine and malrotation. In addition, all patients with CIIP described with FLNA mutations have a congenital short bowel as a common feature. Male patients with CSBS with either missense mutations or distal truncating mutations in FLNA have, in general, multiple congenital anomalies in addition to a congenital shortened small intestine. However, the male patients in our study and the male patient described by Gargiulo et al.10 presented with a gastrointestinal phenotype, including malrotation, a shortened small intestine, and pseudo-obstruction, and they have a 2-bp deletion in the second exon of FLNA. Therefore, starting with screening of exon 2 of FLNA is recommended in male patients presenting with CSBS without mutations in CLMP. If no mutation is found in the second exon of FLNA, screening the entire FLNA gene might reveal another, more distal, nontruncating mutation. In male patients with CSBS and multiple major congenital anomalies, it is worth screening the entire FLNA gene for mutations. A family history of features associated with FLNA mutations, like periventricular nodular heterotopia and cardiac valvular dystrophy, can also point to FLNA mutations. Furthermore, as FLNA mutations have not been found in patients with only pseudo-obstruction, we argue that a diagnosis of CSBS rather than CIIP, with or without central nervous system involvement, should drive additional genetic screening for mutations in FLNA in male patients. These data also suggest that FLNA could be seen as the underlying gene for CSBS rather than for CIIP.10 As mutations in either FLNA or CLMP can explain CSBS, further research is needed to understand a possible interaction of their gene products.
We do not know whether FLNA and CLMP are directly linked or are part of the same protein network. Raschperger et al.19 showed that CLMP co-localizes with actin filaments. They speculated that CLMP interacts with a protein that directly binds to actin filaments, which would bring CLMP to the tight junction by anchoring CLMP in the actin cytoskeleton. They suggested that zonula occludens-1 (ZO-1) could be such an interacting protein. As FLNA also binds to actin filaments, we can speculate that FLNA is the link between CLMP and the actin cytoskeleton. It is known that FLNA interacts with other transmembrane proteins such as integrin beta and the cystic fibrosis transmembrane conductance regulator.20 There is also supportive evidence that FLNA plays a role in anchoring transmembrane proteins in the cell membrane. It has been shown, e.g., that for the expression of cystic fibrosis transmembrane conductance regulator on the cell membrane, its interaction with FLNA is important.20 Therefore, we might suspect that FLNA plays a role in the internalization of CLMP in the plasma membrane as well. It can be speculated that the mutations in FLNA influence the expression of CLMP on the plasma membrane. Further research is needed to determine whether CLMP and FLNA do indeed interact in the same protein network and, if so, which other proteins are involved in this network. We cannot exclude that different pathways underlie X-linked CSBS and autosomal recessive CSBS, with different disease mechanisms leading to a similar disease phenotype.
The authors declare no conflict of interest.
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This work was funded by the Junior Scientific Masterclass (University of Groningen) and the J.K. de Cock Foundation. We thank the patients and their families for participating in this study, Jackie Senior for editing the manuscript, and Dineke S. Verbeek for critically reviewing the manuscript. C.S.v.d.W. and R.M.W.H. conceptualized and designed the study. C.S.v.d.W., Y.S., J.B.G.M.V., M.C., E.O., C.-H.C., A.S.B., M.K.L., J.P.A., and R.M.W.H. generated, collected, assembled, analyzed, and/or interpreted the data. C.S.v.d.W. and R.M.W.H. drafted and revised the manuscript. R.M.W.H. approved the final version of the manuscript.
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van der Werf, C., Sribudiani, Y., Verheij, J. et al. Congenital short bowel syndrome as the presenting symptom in male patients with FLNA mutations. Genet Med 15, 310–313 (2013). https://doi.org/10.1038/gim.2012.123
- chronic idiopathic intestinal pseudo-obstruction
- short small intestine
- synovial lipomatosis
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