Molecular Analysis of Berardinelli-Seip Congenital Lipodystrophy in Oman: Evidence for Multiple Loci

Congenital generalized lipodystrophy (CGL; Berardinelli-Seip syndrome MIM 269700) is a rare disorder that is autosomal recessive in inheritance. CGL is characterized by the almost complete absence of body fat deposition and is usually evident at birth or during early infancy. In addition, the syndrome often includes the following features: organomegaly, muscle hypertrophy, acanthosis nigricans, hyperandrogenism, hyperlipidemia, and hyperinsulinemia or insulin-resistant diabetes. Garg et al. (1) mapped a locus for CGL to 9q34 (BSCL1), a 9-cM interval between markers D9S1818 and D9S1826, but the gene is not yet identified at this locus. Of the 17 pedigrees analyzed, 2 showed no linkage to this locus, suggesting that CGL was genetically heterogeneous. Recently, CGL (BSCL2) in nine pedigrees from two geographical clusters, Lebanon and Norway, was mapped to 11q13 between markers D11S4076 and D11S480 (2). Magré et al. (2) identified a gene, designated the seipin gene, within this locus and demonstrated functional mutations within this gene in their CGL cohort. Twenty additional families of various ethnicities were analyzed, and the disease was found to cosegregate with either 9q34 or 11q13.

We have identified 16 subjects with CGL from 11 consanguineous sibships in Oman originating from three different geographical areas (groups A, B, and C) (Fig. 1). The evaluation of the main clinical and biological characteristics of subjects revealed no obvious differences between subjects in groups A and B, but a variable expression of the disease in group C. Affected individuals in group C had lipodystrophy and muscle hypertrophy as common features but did not show significant developmental delay, acanthosis nigricans, or childhood-onset insulin resistance. In addition, all individuals in group C and none in groups A and B were affected by cardiomegaly and hypertrophic pyloric stenosis. Group C also showed reduced exercise tolerance, percussion myoedema in skeletal muscle (the production of a dimple in the muscle after pressing on the surface), and disturbance of cardiac rhythm and cardiomyopathy. Detailed clinical findings are described elsewhere (3). As all subjects were the offspring of consanguineous marriages, we decided to use a homozygosity mapping approach to investigate the genes involved in these forms of CGL. This approach was based on the premise that in the event of a rare recessive disease, the affected progeny of a consanguineous mating will have inherited two identical copies of the disease gene from a common ancestor and therefore should be homozygous for the defective allele.

The 11q13 locus was initially investigated for homozygosity using microsatellite markers D11S4191, D11S4076, CA10, CA9, D11S1883, and PYGM and five SNPs within BSCL2. In group A, subjects A1, A2, A3, and A4 were homozygous for the microsatellites flanking BSCL2 and the SNPs within the gene. These families were examined for additional microsatellites at the BSCL2 locus to determine the extent of the homozygosity at this locus (Table 1). For A3, the flanking homozygous markers are CA10 and D11S4113, which are about 6.5 cM apart. This is of interest when considering the BSCL1 locus in these families (see below). Subject A4 was homozygous for the microsatellites and for the five single nucleotide polymorphisms (SNPs) listed in Table 1 but was found to be heterozygous for an SNP in intron 1 [1 (+62 → +64) GGG/deletionGGG]. A5 was homozygous for the five seipin SNPs, as were both parents, and A5 was heterozygous for the microsatellites D11S4076–D11S1883 (Table 1). Subject A4 had a small homozygous interval, but the internal polymorphism in the seipin gene indicated that the homozygosity in this subject was probably coincidental and not indicative of the presence of a causative gene. No mutation was found in this family, but the possibility exists that the intron 1 polymorphism was a de novo event, and there was a noncoding mutation in the seipin gene in this pedigree, as the mutation screen (see below) did not cover noncoding regions.

Mutation analysis of the seipin gene in all affected individuals revealed that subjects from families A1 and A3 had a novel homozygous mutation in exon 7 that would give rise to a stop codon (W259X). Subject A2 had a novel mutation in the acceptor splice site of intron 6 (cDNA:1016/-2 A→C). A mutation previously found near this splice site (cDNA:1016/-3) in a family originating from Turkey was demonstrated to cause skipping of the next exon, exon 7 (2). Therefore, it is likely that the mutation we identified at the adjacent base, which belongs to a strictly conserved sequence for RNA splicing, causes exon 7 to be skipped. No mutations were detected in the other samples from group A. It is interesting to note that although these subjects, A1, A2, and A3, reside in the same geographic area of Oman, two different mutations within the seipin gene were observed, suggesting that these individuals do not share a common ancestor from whom the lipodystrophy-causing allele was inherited.

Moreover, although all families from group A are from the same area in Oman and patients present with a similar phenotype, at least two loci are involved. In families from groups B and C, haplotype analysis indicated that the disease was not linked to BSCL2, as all sibships showed heterozygosity for the interval examined on chromosome 11. No mutations were detected in any of these samples on analysis of the seipin gene.

Homozygosity at markers flanking the 9q34 locus was investigated in the Omani families and is shown in Table 2. It was found that subjects A4, A5, C1, C2, C3, and C4 could map to this locus. However, the majority of subjects were only homozygous for either D9S1818 or D9S1826, and in subject C4, who was homozygous for both markers, this is uninformative because her mother and unaffected brother were similarly homozygous. The distance between these microsatellites at the 9q34 locus is 9 cM; thus, it is possible that recombination could have occurred within this chromosomal region, especially when considering the smaller interval found at the BSCL2 locus for A3, as described above. Unfortunately, no microsatellite markers have been identified between D9S1818 and D9S1826 that could be used to obtain further mapping information; therefore, only the discovery of new markers internal to the existing interval will enable further analysis of this locus. Neither family C5 nor B1 were compatible with linkage to BSCL1. The similarity in phenotype of Group C does not make it unreasonable to assume that the BSCL1 locus may not be involved in any of these subjects, but such an assumption is not certain. Even if this is so and CGL in all individuals in Group C is produced by mutations in an unidentified gene, family B1 appears unlikely, by virtue of its phenotype, to map to the same locus.

Other candidates for the lipodystrophy locus in the Omani subjects have been considered. Using the ABI LMS v2 markers, homozygosity mapping has been performed on chromosomes 1q, 2p, 18p, and 20q, the locations of LMNA, LPIN1, LPIN2, and LPIN3, respectively. Mutations in LMNA have previously been reported in subjects of autosomal dominant familial partial lipodystrophy (Dunnigan type) (4–6) but not in CGL patients (7). The LPIN genes were considered because lipodystrophy is a component of the fld mouse model phenotype; the fld mouse was demonstrated to have a mutation in the mouse Lpin1 gene that is highly expressed in adipose tissue (8). We examined linkage to these loci in all samples and neither sibship B1 nor C5 demonstrated homozygosity at these loci.

In conclusion, novel mutations in the seipin gene (W259X and cDNA:1,016/-2 A→C) were identified in three of five CGL subjects in group A of the Omani samples. Interestingly, the haplotypes and mutations observed in these subjects were not identical, suggesting that although group A shared a similar phenotype and the same geographical origin, the lipodystrophy-associated locus was not inherited from a common ancestor. Group B and C subjects appear to be unlinked to BSCL2. The majority of subjects unlinked to 11q13 showed homozygosity at one flanking marker of the 9q34 locus. As the interval between these markers is large, we cannot exclude the possibility that subjects A4, A5, C1, C2, C3, and C4 were linked to this locus and underwent recombination within the interval. However, the shared and distinct phenotype of all families in group C strongly suggests that they may share a common genetic etiology that is distinct from family B1. It therefore appears that there are two or more novel loci for autosomal recessive CGL in the Omani population.