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Oct 25, 1996 - history of osteopetrosis, renal tubular acidosis, mental re- tardation, and CA II deficiency were studied to test the hypothesis that the mutation, ...

Hum Genet (1997) 99 : 634–637

© Springer-Verlag 1997

O R I G I N A L I N V E S T I G AT I O N

Dahmani M. Fathallah · Mohamed Bejaoui · Denis Lepaslier · Khelifa Chater · William S. Sly · Koussay Dellagi

Carbonic anhydrase II (CA II) deficiency in Maghrebian patients: evidence for founder effect and genomic recombination at the CA II locus

Received: 5 July 1996 / Revised: 25 October 1996

Abstract A splice junction mutation at the exon 2 – intron 2 boundary of the carbonic anhydrase II (CA II) gene was previously shown to be the unique mutation underlying the CA II deficiency syndrome in patients of Arab descent. Fourteen Tunisian (Maghrebian) families with a history of osteopetrosis, renal tubular acidosis, mental retardation, and CA II deficiency were studied to test the hypothesis that the mutation, found in all 24 patients, derived from a common ancestor originating in the Arabic Peninsula. A filiation study permitted us to trace these families back to a common Arabic tribe that settled in the Maghreb in the tenth century, indicating a common ethnic origin for these families. Segregation of the mutation with a TaqI biallelic restriction site polymorphism upstream of the CA II gene was studied by sequence-tagged site analysis in all the family members. These studies showed cosegregation of the Taq (-) allele with the mutation in 12 families out of 14. This observation supports a founder effect to explain the common CA II deficiency allele in this population. In the remaining two families, a genomic recombination or gene conversion occurred between the TaqI restriction marker and the mutation causing the disease. The relatively high recombination frequency sug-

D. M. Fathallah (Y) · K. Dellagi Human Molecular Genetics Group, Laboratory of Immunology, Institute Pasteur of Tunis, P.O. Box 74-1002 le Belvedere, Tunis, Tunisia Tel.: + 216-1-789 608; Fax: + 216-1-791 833 M. Bejaoui Department of Pediatrics, Charles Nicolle Hospital, Tunis, Tunisia D. Lepaslier Centre d’Etude du Polymorphisme Humain CEPH, Paris, France K. Chater Department of History, Faculty of Arts and Literature, University of Tunis, Tunisia W. S. Sly Department of Genetics and Molecular Biology, Saint Louis University, St. Louis, USA

gests the presence of a hot spot for recombination or gene conversion at the CA II locus.

Introduction Carbonic anhydrase II (CA II) deficiency is the primary defect in the autosomal, recessively transmitted syndrome of osteopetrosis (defective bone resorption) with renal tubular acidosis, cerebral calcification, and mental retardation (Sly et al. 1983). Although it has been reported in families from various ethnic origins (Belgian, American, French, German, Italian, Hispanic, Japanese), this syndrome is particularly common in Arab populations of the Middle East (Egypt, Saudi Arabia, Kuwait) and the Maghreb (Tunisia, Algeria, Morocco) (reviewed in Whyte 1993). More than 70% of the reported cases are from this part of the world. This striking geographical distribution is probably the result of a characteristically high rate of consanguineous marriages and an increased frequency of the CA II deficiency allele in Arab populations. This allelic distribution agrees with the historically well documented migration of people from the Arabic Peninsula to the Maghreb between the eighth and tenth centuries (Idriss 1971). The human gene encoding CA II has been cloned (Venta et al. 1991) and mapped to chromosome 8q22 (Nakai et al. 1987) within a gene cluster containing genes for CA I and CA III. A linked TaqI restriction fragment length polymorphism has been identified less than 1 kb upstream of the CA II gene (Venta et al. 1983). Mutational studies of CA II deficient patients of different ethnic origins have disclosed molecular genetic heterogeneity underlying the disease (Venta et al. 1991; Roth et al. 1992; Hu et al. 1992). Several mutations have been described, two of which are associated with defined ethnic groups (Fathallah et al. 1994; Hu et al. 1994). Patients of Arabic origin have a unique splice junction mutation at the boundary of exon 2 – intron 2 of the CA II gene. The genetic homogeneity within this group is reflected in many of the clinical manifestations, the Arabic patients

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A B Fig. 1 A Ethnic filiation of the carbonic anhydrase II deficient Tunisian families. The 24 patients belong to 14 families, six of which bear different family names and were presumably unrelated. The search for the ethnic origin showed that families AM, AY, and FR were members of the Ouled Aoun tribe, while families SA, BB, and YH belong to the same tribe of Ouled Mhena (family YH could be traced directly to the older tribe of Ryah). The two intermediate tribes were established in the Maghreb in the sixteenth century AD and were derived from the same tribe of Ryah. This latter was a fraction of the Helal tribe, which is one of the three major Arab tribes (along with the Mahequil and the Esquequin) that effectively settled in the Maghreb in the year 999 AD. The term Ouled means sons of. Historically, an Arab tribe is defined as a large group of people who have strong familial relationship and are all descendants of the same ancestor who gives the tribe his name. B Map of the Maghreb (North Africa) and the Middle East. Arrows indicate the migration flow from the Arabic Peninsula to the Maghreb and Asia. The tribe of Helal, the potential founder of the population we have studied, settled in Egypt before migrating to the Maghreb

being among the most severely affected, and having mental retardation as a constant feature (Bejaoui et al. 1991). In this report we present evidence that the unique mutation underlying the CA II deficiency in 14 multiply affected Tunisian families arose in a common ancestor originating in the Arabic Peninsula. We have identified an ancestral association of a polymorphic TaqI marker with the disease trait. In addition, our findings suggest that genomic rearrangement might occur at a relatively high frequency in the human CA II locus.

Pedigrees Information was obtained through direct contact and multiple interviews of the family members. In two families studied, living members from as many as five generations were available. The common origin of the families was assessed by an ethno-historical investigation based on the families’ oral traditions as well as an extensive literature search using old manuscripts (e.g., Del Marmol Carvajal 1564). The latter described thoroughly the social organization (repertoire of the Berberian and Arab tribes) of the populations which lived in the Maghreb from the tenth century as well as the migration of people from the Arabic Peninsula to the Maghreb. Sequence-tagged site (STS) and mutational analysis Genomic DNA prepared from each of the 24 patients, their parents, and the 32 available family members, as well as 30 non-affected Tunisian adult controls (randomly selected), was subjected to amplification by using two primers flanking the biallelic Taq polymorphism, as described by Venta and Tashian (1990). The splice junction mutation at the boundary of exon 2 – intron 2 of the CA II gene was investigated in the 14 patients not previously reported, as described by Fathallah et al. (1994). Parentage assessment Paternity was established in each family studied by DNA fingerprinting using the variable number tandem repeat probes 33.6 and 33.15 (kindly provided by A. Jeffreys, Leicester, UK), as described elsewhere (Helminen et al. 1988). Software

Materials and methods Mutational analysis of 6 out of the 14 families studied (ten patients) was reported earlier (Fathallah et al. 1994). Several additional members of these families and members of 8 additional families (14 patients) were included in this study. All the patients were born of consanguineous marriages.

Epi info version 5 was used to perform the Yates corrected chisquare test to assess the association between the TaqI marker and the disease trait. Pedraw version 2.2 was used to draw the family trees.

Results We analyzed 14 multiply affected CA II deficient Tunisian families, totalizing 24 patients, for the segregation of a TaqI biallelic marker located upstream of the CA II gene,

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Fig. 2 Segregation of the TaqI alleles with the mutation underlying carbonic anhydrase II deficiency (ACII-) in two Tunisian families. A Two recombination or gene conversion events are depicted in family FR, the first between mother I-2 and her daughter II-3 and the second between father II-7 and his son, patient III-13. B In family OU, the two possible paternal allelic associations are shown. The potential recombination event could be attributed to the patient or her sister according to the actual father’s genotype

and the splice junction mutation at the boundary of exon 2 – intron 2 of the CA II gene, previously shown to be the unique mutation causing CA II deficiency in patients of Arab origin. We identified a common ancestral root through interviews of the family members and comparison with ancient ethnological manuscripts. This filiation study permitted us to trace all the families to the common ancestral tribe of Helal (Fig. 1 A), which migrated from the Arabic Peninsula and settled in the Maghreb in the tenth century (Fig. 1 B), with Helal being the potential ultimate founder. We have also identified six affected Algerian families that were related and shared the same ancestral origin with the families we have studied. However, we could not investigate the patients. Genetic analysis In 12 families (22 patients), analysis of the TaqI (-) marker segregation showed allelic association with CA II deficiency, with no evidence of recombination between the Taq (-) allele and the CA II (-) mutation site. Of the patients, 93% homozygous for the mutation were also homozygous for the Taq (-) allele. This strong linkage disequilibrium (Yates corrected chi-square = 8.67, P value =

0.00323, odds ratio = 0.09, IC95 = 0.01; 0.53) was confirmed by the investigation of extended pedigrees. This allelic association was present in a large majority (84 out of 93) of affected chromosomes. On the other hand, the allele frequency of the TaqI (-) marker in the non-affected Tunisian population was found to be 50%, which is identical to that reported in the Western population (Venta et al. 1983). Families FR and OU These two families were exceptional. Genotypes were established according to the Mendelian rules of inheritance. The segregation of the TaqI (+) and (-) markers with the mutation is presented with the family trees (Fig. 2). The association of the Taq (+) allele with the mutation was observed only in members of these two families with two probable recombination events occurring in family FR between generations I and II and II and III (Fig. 2 A). Another instance of Taq (+), CA II (-) association was found in family OU (Fig. 2 B), but the origin of the recombination/gene conversion event could not be established with certainty. We assessed the paternity in all the families studied by DNA fingerprinting to rule out an eventual mismatching between the children and their biological parents and we did not observe any ambiguity (data not shown). To look for more polymorphic markers that would help extend the study to haplotype analysis, we screened the CEPH YAC library and isolated five contig clones (764D11, 790A7, 790B5, 804D10, and 956C3) that contained the CA I, CA II, and CA III genes (D. Lepaslier and D.M. Fathallah, unpuplished data). Unfortunately,

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none of the available STSs mapping to chromosome 8q22-23 were within the isolated clones.

Discussion Given the fact that the migration of populations from the Arabic Peninsula to the Maghreb is historically well documented and that the frequency of CA II deficiency in Saudi Arabia is elevated (Sly et al. 1985; Cochat et al. 1987; Strisciuglio et al. 1990), we addressed the issue of whether the common and unique mutation underlying CA II deficiency in Tunisian families arose in a common ancestor originating in the Arabic Peninsula. We have conducted a filiation study, demonstrating that all the families we have studied, and probably six related, affected Algerian families, were of Arabic descent and could be traced back to the old tribe of Helal that settled in the Maghreb in the tenth century. We have also analyzed the segregation of a biallelic TaqI marker upstream of the CA II gene in association with the mutation and found that the TaqI (-), CA II (-) allelic association was predominant within the affected families. The presence in the Maghrebian patients of Arab origin of a predominant allelic association including the mutation, supports a founder effect as the basis of the occurrence of the disease in the Maghreb. Furthermore, it suggests that CA II deficiency was introduced into the Maghreb on an ancestral chromosome with the Taq (-) tightly linked marker associated with the disease allele. Since a significant fraction of the Maghrebian population (Tunisian, Algerian, Moroccan) originated in the Middle East, it is likely that this haplotype will also be common in patients from genetically related populations of this part of the world such as Syrian, Yemeni, Iraqi, Palestinian, and Sephardic Jewish. It would be of interest to know whether there is a selective association between one of the TaqI allelic forms and the mutation in patients of other ethnic origins and whether a correlation can be drawn between a given mutation, the TaqI marker, and a clinical phenotype, such as the relatively severe form which includes mental retardation (Fathallah et al., in preparation). The observation of the alternate TaqI allele in association with the mutation in two separate families could be explained either by a recurrent mutation at the TaqI site or by the occurrence of a recombination event or gene conversion in this region of the CA II locus. The apparently high frequency of this event suggests the existence of a hot spot for gene conversion or recombination upstream of the CA II gene. Presumably, the existence of DNA sequences that could serve as a recombination signal (Venta et al. 1985; Shapiro et al. 1987) upstream of the CA II gene would favor the recombination or gene conversion alternative and offers a structural basis to explain the elevated recombination frequency. Acknowledgements This work was supported by a grant from the Tunisian State Secretariat for Scientific Research and Technology. We are grateful to the members of the families for their kind collaboration in the study. We thank Dr. H. Louzir for his help with the art work.

References Bejaoui M, Kamoun A, Baraket M, Bourguiba H, Lakhoua R (1991) Le syndrome associant: osteopetrose, acidose tubulaire, retard mental et calcifications intracraniennes par deficit en anhydrase carbonique II. Arch Pediatr 48 : 711–714 Cochat P, Loras-Duclaux I, Guibaud P (1987) Deficit en anhydrase carbonique II: osteopetrose, acidose renal tubulaire et calcification intercraniennes. Revues de la literature a partir de trois observations. Arch Pediatr 42 : 121–128 Del Marmol Carvajal (1564) Description generale d’Afrique. Availlable at the library of the Institut des Belles Lettres Arabe, Tunis, Tunisia Fathallah DM, Bejaoui M, Sly WS, Lakhoua R, Dellagi K (1994) A unique mutation underlying carbonic anhydrase II deficiency syndrome in patients of Arab descent. Hum Genet 94 : 581–582 Helminen P, Ehnholm C, Lokki ML, Jeffreys A, Peltonen L (1988) Application of DNA “fingerprints” to paternity determinations. Lancet 12 : 574–576 Hu PY, Roth DE, Skaggs LA, Venta PJ, Tashian RE, Guibaud P, Sly WS (1992) A splice junction mutation in intron 2 of the carbonic anhydrase II gene of osteopetrosis patients from Arabic countries. Hum Mutat 1 : 288–292 Hu PY, Ernst AR, Sly WS, Venta PJ, Skaggs LA, Tashian RE (1994) Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean hispanic patients. Am J Hum Genet 54 : 602–608 Idriss HR (1971) Hilal. In: Brill EJ (ed) Encyclopedie de l’islam, T3. Lyde, Paris, pp 398–399 Nakai H, Byers MG, Venta PJ, Tashian RE, Shows TB (1987) The gene for human carbonic anhydrase II (CA 2) is located on chromosome 8q22. Cytogenet Cell Genet 44 : 234–235 Roth DE, Venta PJ, Tashian RE, Sly WS (1992) Molecular basis of human carbonic anhydrase II deficiency. Proc Natl Acad Sci USA 89 : 1804–1808 Shapiro LH, Venta PJ, Tashian RE (1987) Molecular analysis of G+C-rich upstream sequences regulating transcription of the human carbonic anhydrase II gene. Mol Cell Biol 7 : 4589–4593 Sly WS, Hewett-Emmett D, Whyte MP, Yu YL, Tashian RE (1983) Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA) 80 : 2752–2756 Sly WS, Whyte MP, Sundaram V, Tashian RE, Hewett-Emmett D, Guibaud P, Vainsel M, Baluate HJ, Gruskin A, Al-Mosawi M, Sakati N, Ohlsson A (1985) Carbonic anhydrase II deficiency in 12 family with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. N Engl J Med 313 : 139–145 Strisciuglio P, Sartorio R, Pecoraro C, Lotito F, Sly WS (1990) Variable clinical presentation of carbonic anhydrase deficiency: evidence for heterogeneity? Eur J Pediatr 149 : 337–340 Venta PJ, Tashian RE (1990) PCR detection of the TaqI polymorphism at the CA II locus. Nucleic Acids Res 19 : 4795 Venta PJ, Shows THB, Curtis PJ, Tashian RE (1983) Polymorphic gene for human carbonic anhydrase II: a molecular disease marker located on chromosome 8. Proc Natl Acad Sci USA 80 : 4437–4440 Venta PJ, Montgomry JC, Hewett-Emmet D, Tashian RE (1985) Comparison of the 5´ regions of human and mouse carbonic anhydrase II genes and identification of possible regulatory elements. Biochim Biophys Acta 826 : 195–201 Venta PJ, Welty RJ, Johnson TM, Sly WS, Tashian RE (1991) Carbonic anhydrase II deficiency syndrome in a Belgian family is caused by a point mutation at an invariant histidine residue (107 His → Tyr): complete structure of the normal human CA II gene. Am J Hum Genet 49 : 1082–1090 Whyte MP (1993) Carbonic anhydrase II deficiency. Clin Orthop 294 : 52–63

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