Familial Amyloid Polyneuropathy (TTR Ala 60) in J Neurol Neurosurg Psychiatry: First Published As 10.1136/Jnnp.59.1.45 on 1 July 1995
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J7ournal ofNeurology, Neurosurgery, and Psychiatry 1995;59:45-49 45 Familial amyloid polyneuropathy (TTR ala 60) in J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.59.1.45 on 1 July 1995. Downloaded from north west Ireland: a clinical, genetic, and epidemiological study M M Reilly, H Staunton, A E Harding Abstract family from the Appalachian region of the A cluster of cases of familial amyloid United States7 and more recently reported in polyneuropathy has been described in two further American kindreds.89 The Donegal, north west Ireland. Two Appalachian kindred and one of the other patients from this region have been American kindreds are Irish in origin8 (A shown to have the ala 60 mutation in the Koeppen, personal communication); the ori- transthyretin gene. Three kindreds with gin of the third kindred is not known. In the this mutation have also been described in initial report by Staunton and colleagues,5 the United States. Genealogical and hap- only four of the eight patients gave a history of lotype studies indicate that all known affected relatives, and it could not be assumed patients with this mutation are related that all the cases had a genetic basis and if so, and are descended from a founder in whether all the patients had the same disease. north west Ireland. There is evidence for If this were the case, it seemed clear that the reduced penetrance of this disorder. A disorder exhibited reduced penetrance. population based study showed that 1P1% We have re-examined all known families of the population in this area in north with presumed FAP in Donegal and have west Ireland carry the mutation. This has analysed the TTR gene for the ala 60 muta- implications in terms of diagnosis, tion in patients and selected relatives. We genetic counselling, and treatment in the have also attempted to confirm the suspicion future. that all these patients, and those reported in the United States, are descended from a com- mon extended and (7 Neurol Neurosurg Psychiatry 1995;59:45-49) founder by pedigree haplo- type analysis. Finally, we carried out a Keywords: familial amyloid polyneuropathy; amyloid; population based study in the particular area transthyretin of Donegal involved to assess the prevalence of gene carriers among persons not known to Familial amyloid polyneuropathy (FAP) is an be at risk of FAP. autosomal dominant disorder most com- monly caused by the deposition of variant transthyretin (TTR) in affected tissues.' It Patients and methods http://jnnp.bmj.com/ was originally described by Andrade in PATIENTS Portuguese kindreds in 19521 and subse- The details of the index cases of six of the quently the variant TTR responsible for the original families (one index case was illegiti- Portuguese cases was found to have a substi- mate and no further information was avail- tution of methionine for valine at position 30. able) and the more recently described family There are now at least 27 point mutations in were available to us from earlier studies.56 By the TTR gene associated with peripheral neu- contacting and later visiting these families and on September 23, 2021 by guest. Protected copyright. ropathy (as opposed to non-neuropathic amy- their relatives, and also the only remaining liv- University loidosis).4 The commonest clinical features ing patient from earlier studies, we were able Department of are those of a small fibre neuropathy with to extend the pedigrees originally presented.5 6 Clinical Neurology, Institute of Neurology, autonomic dysfunction and cardiomyopathy, We also obtained information from the official Queen Square, with vitreous and renal involvement occurring local marriage register, which extends back to London WClN 3BG, less often. Some mutations have characteris- 1860. All earlier public records relating to UK M M Reilly tic, although not distinguishing, clinical fea- pedigrees in Ireland were destroyed in a fire in A E Harding tures. 1916. Useful information on the origin of sur- Richmond Institute In 1987, Staunton and colleagues names came from a special report on the sur- for Neurology and described a cluster of eight patients with FAP names of Ireland from 190910 and from a Neurosurgery, from seven families in a small area of Donegal book on Ulster surnames.12 Beaumont Hospital, Dublin, Ireland in north west Ireland.5 These cases were origi- To study the penetrance of the TTR ala 60 H Staunton nally thought to have the Portuguese type of gene, we interviewed all the relatives of the Correspondence to: FAP, but certain clinical features, including original patients whom we could trace in Professor A E Harding, University Department of the late age of onset, the predominance of the Ireland and England. With ethics committee Clinical Neurology, Institute cardiomyopathy and a neuropathy affecting approval and informed consent these were vis- of Neurology, Queen Square, London WClN all fibre types, were not typical of that muta- ited by one of us (MMR). A full clinical gen- 3BG, UK. tion. In 1991 two further patients from eral and neurological examination was Received 4 November 1994 Donegal (including a first cousin of one of the performed, with special attention to the car- and in revised form were to the 16 February 1995 original cases) found have TTLR diovascular system and motor and sensory Accepted 24 February 1995 ala 60 mutation,6 originally described in a function in the limbs, and a blood sample was 46 Reilly, Staunton, Harding penetrance study. The screening procedure J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.59.1.45 on 1 July 1995. Downloaded from was the same as that in relatives, consisting of "L. clinical examination and TTR gene analysis. Results were not given to either relatives or A ortnatraw the participants in the population study, and < * (~~~~Carrigart) they gave consent on this basis. (Gweedore) A Derrybeg Aunbegg DNA ANALYSES .11 DNA was extracted from blood by standard A A Burtonport ,A/ Bui methods. The TTR mutation underlying the ala 60 variant creates a new restriction site for the restriction endonuclease Pvu II. Genomic DNA was amplified by polymerase chain reaction (PCR) and primers flanking TTR exon 3 (table 1: primers 718 and 719), using a Hybaid Intelligent heating block and Taq polymerase (Promega). Amplification was carried out for 30 cycles, comprising denatur- ing at 90°C (30 s), annealing at 50°C (30 s), and elongation at 70°C (30 s). Products of PCR were digested with Pvu II (Promega) under conditions recommended by the manu- facturers. The digested products were elec- trophoresed in a 3-2% agarose gel (BCR), stained with ethidium bromide, and visualised with ultraviolet light. There are seven polymorphisms in introns of the TTR gene and five different haplotypes designated I-V have been described.'2 13 A has been Co Donegal Ireland haplotype published using six of the seven TTR polymorphisms for one of the Figure 1 Map ofpart of county Donegal (county hatched on map ofIreland in lower American cases with FAP ala 60 (the one right offigure) showing the distribution of the seven originalfamilies with TTR ala 60 whose ancestry is not clear; Ii S, Sommer SS, related FAP area (open triangles) and the from which the random sample of the at population was selected (to the left of broken line). Modifiedfrom Staunton et all with presentation PNA meeting, Rapallo, 1992) permission. and this corresponds to haplotype I with Yoshioka's nomenclature. 12 We analysed polymorphisms 3, 5, and 6'2 to determine the haplotypes of our patients with TTR ala 60 taken. We only screened relatives over the age and also 49 control subjects from the popula- of 40 as these were the only ones likely to have tion study who did not have the mutation. an asymptomatic neuropathy due to the ala Polymorphism 3 is an A/G polymorphism in 60 TTR mutation in view of the late onset of the second intron of the TTR gene, and poly- http://jnnp.bmj.com/ TTR ala 60 FAP.56 In total we screened 28 morphism 5 and 6 G/C and T/G polymor- relatives. phisms in the third intron. Polymorphisms 3 For the population based prevalence study, and 5 are detected by Msp I and Fnu 4H1 we screened 182 people over the age of 40 restriction, using PCR with primers A5 and who were resident in the area from which all A6 and A3 and A9 (table 1) respectively. the original patients originated (fig 1). These Conditions were as used for the exon 3 were ascertained from people attending local primers except that the annealing temperature on September 23, 2021 by guest. Protected copyright. general practitioner's surgeries and the local was 56°C for polymorphism 3 and 5. The hospital outpatient department who did not PCR products were digested with Msp I have cardiac or neurological symptoms (as (Promega) or Fnu 4HI (New England these could be due to FAP) or a family history Biolabs) respectively. of amyloidosis. This age group was again Polymorphism 6 was detected by mismatch selected so that subjects found to have the PCR using primers A3 and A9 (table 1), the TTR ala 60 gene could be included in the last creating a single mismatch of G for A near the 3' end so that the PCR product contains a new restriction site for Rsa I only when G and not T is present at polymorphism 6. The PCR conditions were as for polymorphism 5, with Table 1 Oligonucleotide primers subsequent digestion with Rsa I (Promega). No Position Sequence PCR primers: Exon 3 719 3785-3804 5'-CTCCATGCGTAACTTAATCC-3' Results Exon 3 718 4359-4340 5'-GCCAGATGTGTTTTGGAATG-3' Haplotype Only two affected patients from the seven pre- primers: viously described families (excluding that of Intron 2 A5 2402-2411 5'-ACGATCACCCGACTTCATGT-3' Intron 2 A6 2563-2543 5'-AAGGGTCTCCTGATTGCTCTG-3' the illegitimate patient) were still alive, one Intron 3 A3 5572-5591 5'-AATGCCTCAClllGGTGACG-3' reported by Staunton et a16 and the other a 64 Intron 3 A9 5733-5714 5'-GAGGCCTAAGAGCTCCTAGT-3' year old man with onset of paraesthesiae and Underlining indicates mismatch site.