Correspondence

Connexin43 Mutations in Sporadic and Familial Defects of Laterality

N Engl J Med 1995; 333:941-942October 5, 1995

Article

To the Editor:

Britz-Cunningham et al. (May 18 issue)1 describe missense base substitutions in the connexin43 gene in all six patients with heterotaxia they studied. Heterotaxia is a syndrome of multiple malformations in which the developing embryo fails to establish normal left–right asymmetry. Four of the six patients carried two different base changes, and one base change, Ser364Pro, was identified in five. The results suggest that connexin43 mutations account for a large percentage of the cases of heterotaxia, that most such cases are likely to be recessive, and that one base substitution, Ser364Pro, may account for a considerable percentage of mutant connexin43 chromosomes.

To test these hypotheses, we amplified and directly sequenced the region of connexin43 that codes for the cytoplasmic tail in 15 patients with sporadic defects of laterality and 3 with familial defects of laterality. All of the nucleotide changes reported by Britz-Cunningham et al. are contained within this portion of the gene. The 15 patients with sporadic defects (7 female and 8 male) had features of heterotaxia, including severe heart malformations in all but 1. One of the affected male patients has a brother with an isolated atrial septal defect. The patients with familial defects of laterality are from kindreds with apparent autosomal dominant transmission of the trait.

No base changes in the coding sequence were detected in any of the patients studied. Specifically, none of the base substitutions reported by Britz-Cunningham et al., including the Ser364Pro mutation, were identified. The insertion of a single base in the 3' untranslated region was identified in the same position in two patients.

Previous studies imply that several genes are involved in establishing normal laterality in mammals.2-5 Although Britz-Cunningham et al. suggest that connexin43 may have a role in that process, the relative contribution of connexin43 mutations to defects of laterality in humans remains to be clarified.

Brett Casey, M.D.
Baylor College of Medicine, Houston, TX 77030-3498

Andrea Ballabio, M.D.
Telethon Institute of Genetics and Medicine, 20132 Milan, Italy

5 References

1. 1

   Britz-Cunningham SH, Shah MM, Zuppan CW, Fletcher WH. Mutations of the connexin43 gap-junction gene in patients with heart malformations and defects of laterality. N Engl J Med 1995;332:1323-1329
   Full Text | Web of Science | Medline

2. 2

   Brueckner M, McGrath J, D'Eustachio P, Horwich AL. Establishment of left-right asymmetry in vertebrates: genetically distinct steps are involved. In: Bock GR, Marsh J, eds. Biological asymmetry and handedness. Ciba Foundation Symposium 162. Chichester, United Kingdom: John Wiley, 1991:202-18.
   

3. 3

   Burn J. Disturbance of morphological laterality in humans. In: Bock GR, Marsh J, eds. Biological asymmetry and handedness. Ciba Foundation Symposium 162. Chichester, United Kingdom: John Wiley, 1991:282-99.
   

4. 4

   Casey B, Devoto M, Jones KL, Ballabio A. Mapping a gene for familial situs abnormalities to human chromosome Xq24-q27.1. Nat Genet 1993;5:403-407
   CrossRef | Web of Science | Medline

5. 5

   Alonso S, Pierpont ME, Radtke W, et al. Heterotaxia syndrome and autosomal dominant inheritance. Am J Med Genet 1995;56:12-15
   CrossRef | Web of Science | Medline

To the Editor:

Britz-Cunningham et al. identified single-base substitutions in the cytoplasmic tail of the connexin43 gene in patients with heart malformations and defects of laterality. A defect in a protein involved in cell–cell communication in patients with laterality disturbances is in keeping with current models of the determination of left–right asymmetry.1 Their finding of mutations in all six patients with visceroatrial heterotaxia studied is surprising, given the evidence of genetic heterogeneity in this condition.

The mutations detected by Britz-Cunningham et al. were in codons 364, 352, 365, 373, and 326. In four patients, two mutations were identified, suggesting an autosomal recessive defect. To date we have sequenced the terminal 500 base pairs of the connexin43 gene coding region in 12 patients with defects of laterality and have not detected mutations in the above-mentioned codons or any other mutations. The patients all had visceroatrial heterotaxia with a complex heart defect, including one with asplenia syndrome and right atrial isomerism and nine with left atrial isomerism, of whom four had known polysplenia. one patient had an affected sibling, and 11 had no known affected relatives. Five were from an inbred Pakistani population and had consanguineous parents, making an autosomal recessive defect likely; the remaining seven were non-Pakistani whites. We do not think that our patients are clinically distinct from the Loma Linda patients despite their selection on the basis of heart transplantation.

We studied genomic DNA from whole blood. It is not clear whether Britz-Cunningham et al. detected the mutations in any tissue other than heart. If they confined themselves to cardiac tissue it is possible that their findings represent somatic rather than germ-line mutations. The question could easily be resolved by studying parental DNA.

Miranda Penman Splitt, M.B., B.S.
John Burn, M.D.
Judith Goodship, M.D.
University of Newcastle upon Tyne, Newcastle NE2 4AA, United Kingdom

1 References

1. 1

   Almirantis Y. Left-right asymmetry in vertebrates. Bioessays 1995;17:79-83
   CrossRef | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: Drs. Casey and Ballabio and Splitt et al. should be congratulated for attempting to repeat our so recently published study. As they point out, many genes are likely to be involved in the determination of normal laterality. Knowing this, we did not imply, nor do we believe, that connexin43 mutations, or more specifically, the Ser364Pro substitution, will account for all or even a large fraction of visceroatrial heterotaxia.

On the basis of several years of preliminary study1,2 and emerging embryologic evidence,3 we hypothesized that mutations of connexin43 that affect sites regulating the gap-junction channels formed by the connexin43 protein could result in developmental or functional anomalies of the heart. This view appears to be supported by our results.

It is not clear to us why our children with visceroatrial heterotaxia should differ from those studied by the corresponding authors. However, as stated in our report, the connexin43 gene defects may define a subtype of visceroatrial heterotaxia. In this regard, it is notable that all but one of the children with a Ser364Pro substitution had polysplenia or asplenia and either pulmonary atresia or stenosis. The latter two features may be important in view of the fact that pulmonary atresia has been consistently found in mice with a connexin43 gene “knockout.” 4 In addition, the formation of the pulmonary outflow tract involves neural-crest tissue, which expresses high levels of connexin43.3 Splitt et al. did not describe pulmonary atresia or stenosis in their patients.

Although we cannot completely rule out the possibility of somatic mutation, preliminary results show that a parent of one of the children with visceroatrial heterotaxia has a Ser364Pro substitution. We are currently examining available parents and siblings of the children described in our report, as well as specimens from throughout North America, in the hope of understanding the frequency, penetrance, and expressivity of connexin43 mutations in patients with visceroatrial heterotaxia. Clearly, additional molecular genetic studies of all types of patients with visceroatrial heterotaxia are needed. The determination of laterality during organogenesis is poorly understood and almost certainly complex. Exactly how mutations of the connexin43 gene are involved in this process remains to be seen; however, we believe the results we reported give important clues about the timing and sites of early events in the development of visceroatrial heterotaxia.

William H. Fletcher, Ph.D.
Scott H. Britz-Cunningham, B.S.
Craig W. Zuppan, M.D.
Loma Linda University, Loma Linda, CA 92354

4 References

1. 1

   Stagg RB, Fletcher WH. The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation. Endocr Rev 1990;11:302-325
   CrossRef | Web of Science | Medline

2. 2

   Godwin AJ, Green LM, Walsh MP, McDonald JR, Walsh DA, Fletcher WH. In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C. Mol Cell Biochem 1993;127:293-307
   CrossRef | Medline

3. 3

   Ruangvoravat CP, Lo CW. Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains. Dev Dyn 1992;194:261-281
   CrossRef | Web of Science | Medline

4. 4

   Reaume A, de Sousa PA, Kulkarni S, et al. Cardiac malformation in neonatal mice lacking connexin43. Science 1995;267:1831-1834
   CrossRef | Web of Science | Medline

Citing Articles (12)

Citing Articles

1. 1

   Laura N. Vandenberg, Michael Levin. (2010) Far from solved: A perspective on what we know about early mechanisms of left-right asymmetry. Developmental Dynamics 239:12, 3131-3146
   CrossRef

2. 2

   Alessandro De Luca, Anna Sarkozy, Federica Consoli, Andrea De Zorzi, Rita Mingarelli, Maria Cristina Digilio, Bruno Marino, Bruno Dallapiccola. (2010) Exclusion of Cx43 gene mutation as a major cause of criss-cross heart anomaly in man. International Journal of Cardiology 144:2, 300-302
   CrossRef

3. 3

   LI WANG, GANG LI, JINHONG WANG, SHAOHUI YE, GARETH JONES, SHUYI ZHANG. (2009) Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera). Genetics Research 91:02, 101
   CrossRef

4. 4

   Chih-Jen Wei, Xin Xu, Cecilia W. Lo. (2004) CONNEXINS AND CELL SIGNALING IN DEVELOPMENT AND DISEASE ¹. Annual Review of Cell and Developmental Biology 20:1, 811-838
   CrossRef

5. 5

   K Maclean, SL Dunwoodie. (2004) Breaking symmetry: a clinical overview of left-right patterning. Clinical Genetics 65:6, 441-457
   CrossRef

6. 6

   Vladimir Krutovskikh, Hiroshi Yamasaki. (2000) Connexin gene mutations in human genetic diseases. Mutation Research/Reviews in Mutation Research 462:2-3, 197-207
   CrossRef

7. 7

   Rumiko Kato, Naomichi Matsumoto, Masahiro Fujimoto, Motoi Nakano, Yusuke Nakamura, Norio Niikawa. (1997) Fish mapping of a translocation breakpoint at 6q21 (or q22) in a patient with heterotaxia. The Japanese Journal of Human Genetics 42:4, 525-532
   CrossRef

8. 8

   GREGORY E. MORLEY, JOSÉ F EK-VITORÍN, STEVEN M. TAFFET, MARIO DELMAR. (1997) Structure of Connexin43 and its Regulation by pH _i. Journal of Cardiovascular Electrophysiology 8:8, 939-951
   CrossRef

9. 9

   Rumiko Kato, Naomichi Matsumoto, Norio Niikawa. (1997) Assignment of the human connexin43 gene,GJA1, to chromosome 6q22.3. The Japanese Journal of Human Genetics 42:1, 213-216
   CrossRef

10. 10

    Daniel B. Gros, Habo J. Jongsma. (1996) Connexins in mammalian heart function. BioEssays 18:9, 719-730
    CrossRef

11. 11

    Roberto Bruzzone, Thomas W. White, Daniel A. Goodenough. (1996) The cellular internet: On-line with connexins. BioEssays 18:9, 709-718
    CrossRef

12. 12

    John Burn, Judith Goodship. (1996) Developmental genetics of the heart. Current Opinion in Genetics & Development 6:3, 322-325
    CrossRef