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Reduction of the genetic interval for lymphoedema-distichiasis to below 2 Mb

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Editor—Primary lymphoedema (MIM 153200) is a chronic tissue swelling, most frequently of the lower limbs, which occurs as a consequence of a failure of lymph drainage.1 It arises from an intrinsic abnormality of the lymphatic system and generally shows an autosomal dominant pattern of inheritance with reduced penetrance, variable expression, and variable age of onset.2 There is a strong genetic input into primary lymphoedema, with 35% of all patients showing a positive family history.3 4 The swelling can be present at birth, as in Milroy disease, but more commonly it becomes clinically apparent during puberty, which is known as Meige disease.5-7

A variant of pubertal onset lymphoedema is lymphoedema-distichiasis (LD) (MIM 153400). This syndrome is a rare form of primary lymphoedema which is associated with distichiasis, a congenital anomaly in which aberrant eyelashes arise inappropriately from the site of the meibomian gland openings.4 8 The disease shows an autosomal dominant pattern of inheritance with incomplete penetrance.9 A recent study of three families by our group reported linkage of LD to chromosome 16q24.3.10 The locus was placed between the markers D16S422 and D16S3074, a distance of ∼16 cM according to the Généthon sex averaged map.

Subsequently, more members of family 3 have been ascertained (fig 1) along with an additional family with an affected father and three affected children (not shown). All subjects were carefully phenotyped based on the presence of distichiasis, which provides a clear criterion with which to define affected status. This was established following slit lamp examination by an ophthalmologist unaware of any other clinical signs. DNA of the newly ascertained subjects was extracted from peripheral venous blood by a standard procedure using a Nucleon genomic DNA extraction kit (Nucleon Biosciences, Strathclyde, UK). Polymorphic microsatellite markers were PCR amplified and electrophoresed through an 8% denaturing polyacrylamide gel on a conventional gel rig. Gels were run at a constant 50 mA and the DNA bands were visualised with silver staining.11

Figure 1

Pedigree of family 3 with haplotypes for the markers shown. Newly ascertained subjects are numbered 12–13 in generation II, 13–16 in generation III, and 6–13 in generation IV. All other family members are numbered as they appeared in the initial report.10

The microsatellite markers D16S511, D16S422, D16S402, D16S3037, D16S520, and D16S3074 were PCR amplified in the newly ascertained subjects from family 3 and for the new family. The latter was consistent with linkage to the LD locus. All newly ascertained subjects from family 3 were consistent with linkage, using distichiasis as the sign of affected status. Fig 1 shows the expanded pedigree, with the same numbering for the previously typed subjects as in our original publication.10 It is of interest that IV.5 (fig 1), who was assumed to be unaffected and non-penetrant in the previous study, was found by slit lamp examination to have limited distichiasis. There are therefore no members of any of the four families we have studied who carry the “affected” haplotype but have no signs.

Analysis of an additional six markers within the LD locus, D16S486, D16S498, D16S543, D16S2625, D16S539, and D16S3061, produced a reduction in the distal interval by ∼2 cM as a result of a recombination event in an affected family member (fig 1, IV.4). Another subject (III.5) was included as of unknown status in the initial report. He had a slightly abnormal lymphoscintigraphy result at 30 minutes, which was well within normal limits at 60 minutes, but no lymphoedema. Recent examination has shown him to have no trace of distichiasis, which is unlike all the other carriers of the affected haplotype in the four families analysed. We therefore concluded that he is clinically unaffected and does not carry the mutated gene, rather than being a case of non-penetrance. Analysis of the additional six markers in this subject showed a recombination proximally which removed ∼10 cM from the locus. The flanking recombinant markers are now D16S3037 (proximal) and D16S498 (distal), with D16S520 and D16S486 non-recombinant between these two, which is a genetic distance of ∼4 cM according to the Généthon sex averaged map. Mapping these markers on a mouse/human somatic cell panel12 locates the flanking microsatellite markers between the breakpoints in the hybrids CY120 to CY112. This suggests that the physical distance is 1-2 Mb.

This interval is not very gene rich or physically well characterised, although it does contain the homologue of the Drosophilatranscription factor gene FKHL5. Sequence analysis of BAC RP11-463O9 (AC009108) shows it to contain both D16S520 andFKHL5, placing the gene clearly in the critical region. However, initial analysis of this gene by single stranded conformational polymorphism (SSCP) analysis has shown no mutations in its two exons.

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