PATIENTS AND METHODS

Twenty-one unrelated European patients with the clinical diagnosis of BBS were used in the study. The diagnosis was based on the presence of at least three of the major features of BBS (obesity, retinal dystrophy, polydactyly, renal malformations, mental retardation, and hypogonadism). Genomic DNA was prepared from peripheral blood by a standard salting out procedure.

Mutation screening was performed for BBS1, BBS2, BBS4, and MKKS. The analysis was done by polymerase chain reaction (PCR) amplification and sequencing of genomic DNA. A complete list of primers used for PCR and sequencing is available from the authors.

A PCR protocol was carried out using 100 ng DNA and 10 pmol of each primer in a standard 50 μl reaction. The profile used for amplification in a GeneAmp 2400 PCR cycler was two minutes at 94°C, 37 cycles at 94°C for 15 seconds, 48°C for 30 seconds, 72°C for 30 seconds, and a final extension step at 72°C for two minutes. Direct sequencing of PCR products was carried out using the ABI Prism BigDye Terminator Cycle Sequencing ready reaction kit (Applied Biosystems) and an ABI automated DNA sequencer 310 (Applied Biosystems).

RESULTS AND DISCUSSION

Direct sequencing of the BBS1 gene in 21 patients led to the identification of mutations in five (24%) (table 1). This confirms that mutations in BBS1 account for most cases of BBS among the six mapped loci. Our finding is comparable with a report that linked mutations in BBS1 to 32% of patients with BBS.¹⁶ The previously described M390R mutation was found in all patients on at least one allele (allele frequency 0.14). Among North American patients an allele frequency of 0.32 was found.⁹ One patient was heterozygous for the L518P mutation.

Two new splice site mutations in BBS1 were identified at the splice donor site at the end of exon 9 and 10, respectively. Both mutations were found in affected patients together with the M390R mutation.

All available family members were included in the analysis. Consistent with an autosomal recessive model of inheritance, unaffected family members of three patients were either heterozygous for a mutant allele or had wild type alleles. An affected sister of one patient showed the same mutations as her sib.

In BBS2, two novel mutations were found and in both cases only one mutant allele was identified. The two mutations were R643H and R413X. One of the mutations introduces a stop codon, the other one changes a highly conserved amino acid (table 2) and was not found among 60 controls. The R643H mutation occurred in combination with the sequence change K46R in BBS4. K46R was suggested to be a polymorphism.¹⁶ The other patient was a compound heterozygote for R143X (BBS2) and P503L (BBS4). P503L is a highly conserved residue and was not found in 60 controls. Eight out of 21 patients had the I123V polymorphism. In BBS4, two polymorphisms were found: V284A and T354I.¹⁶

In MKKS two novel mutations were found, a homozygous A181P mutation and a heterozygous D492N mutation. Both mutations occur at highly conserved residues and were not present among 60 healthy controls. No second mutation was identified in the patient with the D492N mutation. In two patients, two polymorphisms (R517C and G532V) were found in MKKS.¹⁶

Among our patients we cannot find support for the hypothesis that BBS1 is involved in triallelic inheritance. The disease segregated as an autosomal recessive disorder including the duplex family. Although we cannot rule out the possibility that the patients have a mutation in one of the unidentified genes (BBS3 or BBS5), this would be unlikely as the remaining genes account only for a very small proportion.¹ Another recent report came to a similar conclusion.¹⁶ Because of the relatively few patients, this study may have missed (rare) cases where BBS1 is involved in triallelism. Another recent study indicates that BBS1 can participate in triallelic inheritance but that other BBS loci, especially BBS2 and BBS6, participate more often in this mode of inheritance.¹⁷

Apart from the five patients with mutations in BBS1, four other patients harboured mutations in BBS2, BBS4, or MKKS. However, in three of these patients, two mutant alleles were not present in the same gene. Two patients had one mutant allele in two genes (BBS2 and BBS4), suggesting a digenic diallelic mode of inheritance. If in one of these cases the K46R (BBS4) mutation is considered a non-disease causing sequence change, then this patient would harbour only one mutant allele in four BBS genes. Another patient was identified with only one mutant allele in MKKS. The detection of only one mutant allele was reported in 10 cases in MKKS.¹⁵ One possibility is that additional mutations were not detected with the methodology used. These mutations would include changes in the promoter region, in introns, or in additional exons. But additonal unidentified mutations would not account for the fact that patients were found with two mutations in two different genes associated with BBS. Although these data do not support triallelic inheritance per se, they strongly support multiallelic inheritance. It provides good evidence for digenic diallelic inheritance in BBS. This phenomenon would also be expected if the three allele hypothesis were correct. Particularly, the patient with the two significant mutations R143X (BBS2) and P503L (BBS4) would be a strong indication for diallelic digenic inheritance.

Another interesting finding is the relatively high percentage of patients with no mutations. At least one mutation was found in nine out of 21 patients (43%). The two remaining mapped loci (BBS3 and BBS5) should only account for a very small percentage of cases. Either a large number of cryptic mutations are present in the five known genes or additional unidentified loci are mutated in most cases.

There is increasing evidence that BBS is involved in a complex mode of inheritance. This is confirmed by the results of our study, which support a digenic mode of inheritance. Experiments of gene interactions will be required to understand the exact mechanism of the disease.