Novel Transthyretin Missense Mutation (Thr34) inan Italian Family With Hereditary Amyloidosis

M. Cristina Patrosso,1 Fabrizio Salvi,2 Domenico De Grandis,3 Paolo Vezzoni,1 Daniel R. Jacobson,4and Alessandra Ferlini1,5*1Istituto di Technologie Biomediche Avanzate, CNR Milano, Italy2Divisione di Neurologia, Ospedale Bellaria, Bologna, Italy3Divisione di Neurologia, Arcispedale S. Anna, Ferrara, Italy4Department of Veterans Affairs Medical Center and New York University, New York, New York5Neuromuscular Unit, Department of Paediatrics & Neonatal Medicine, Hammersmith Hospital,London, United Kingdom

We report on the genetic and molecularcharacterisation of an Italian family with alate-onset, autosomal dominant transthyre-tin amyloidosis. The transthyretin gene wasanalysed by polymerase chain reaction(PCR), restriction generating PCR, and se-quencing, allowing us to discover in one al-lele a novel point mutation. It consists of a Gto C transversion at position 1692 of the ge-nomic sequence, leading to a Thr for Argsubstitution at the position 34 of the poly-peptidic chain. This mutation is associatedwith a severe sensory-motor peripheral neu-ropathy and a restrictive cardiomyopathy.Am. J. Med. Genet. 77:135–138, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: transthyretin; amyloidosis;missense mutation

INTRODUCTION

More than 50 point mutations in the transthyretingene (TTR, MIM *176300) determining single aminoacid substitutions in the protein have been reported sofar [McKusick, 1994; Benson and Uemichi, 1996]. Mostof these TTR variants are amyloidogenic and causeTTR-related autosomal dominant hereditary amyloi-dosis (TTR-HA), although some mutations do not seemto be pathogenic or cause euthyroidotic hyperthyrox-inemia only [Benson, 1995]. TTR-HA represents agroup of autosomal dominant diseases with differentclinical manifestations. Among the reported mutations,

most lead to polyneuropathy and cause familial amy-loidotic polyneuropathy (FAP) [Saraiva, 1991], butsome do not involve the peripheral nervous system,mainly affecting other organs such as myocardium andvitreous body [Benson, 1995]. In rare cases, the diseasehas been reported in homozygotes, with no clear differ-ences in the clinical picture when compared to the het-erozygotes for the same mutations [Jacobson et al.,1997a; Holmgren et al., 1992; Ferlini et al., 1996]. Wereport here clinical and molecular characterisation of anovel TTR missense mutation which co-segregateswith autosomal dominant hereditary amyloidosis in anItalian family. The main clinical findings in our pa-tients consist of sensory-motor peripheral neuropathyand restrictive cardiomyopathy, confirming the obser-vation that TTR mutations are very often associatedwith cardiac involvement.

CLINICAL REPORTS

The family (Fig. 1A) originated from Puglia, a South-ern region of Italy.

Individual I-1 died at the age of 67 years with gen-eralized muscle atrophy and pain of the limbs. No otherclinical data are available.

Individual II-3 presented at the age of 54 years witha sensory-motor polyneuropathy confirmed by EMG.EKG showed a pseudoinfarct pattern and low voltageQRS complex. Echocardiography demonstrated restric-tive cardiomyopathy. The ocular fundi were normal.The patient died at the age of 61 from cardiovascularcomplications.

Individual II-4 was referred to neurosurgery for dor-sal myelopathy (ataxic paraparesis) due to a herniateddisk at the age of 62 years. He presented at the sametime with mild clinical and electromyographic signs ofpolyneuropathy. After surgery the clinical signs of my-elopathy resolved partially. The symptoms related tothe polyneuropathy persisted unchanged. No signs ofcardiomyopathy or vitreous opacities were found.

Individual II-6 presented at the age of 54 years withmuscle cramps and restless leg syndrome. At the age of55 he developed sensory polyneuropathy in the lower

Contract grant sponsor: Telethon-Italy; Contract grant num-ber: 431.

*Correspondence to: Alessandra Ferlini, M.D., NeuromuscularUnit, Department of Paediatrics & Neonatal Medicine, Hammer-smith Hospital, DuCane Road, W12 0NN London, UK. E-mail:aferlini@rpms.ac.uk

Received 17 October 1997; Accepted 29 December 1997

American Journal of Medical Genetics 77:135–138 (1998)

© 1998 Wiley-Liss, Inc.

limbs, confirmed by EMG studies. An EKG showed lowvoltage QRS and left bundle branch block. Echocardi-ography documented restrictive cardiomyopathy. Cur-rently he complains of symptoms related to autonomicneuropathy.

Individual II-7 has suffered from cramps since age of49 years. At age 50 he had signs of sensory polyneu-ropathy. EMG performed at the age of 51 displayed asensory-motor polyneuropathy. EKG showed low volt-age QRS, and echocardiography demonstrated a re-strictive cardiomyopathy. No details of individuals II-1,II-2, and II-5 are available. The individuals in the thirdgeneration, all of them teenagers, are healthy.

MOLECULAR STUDIES

DNA from patients was extracted from peripheralblood using standard procedures. Exons 2–4 of the TTRgene were amplified by polymerase chain reaction(PCR) using 3 pairs of 20–22 base oligonucleotides asprimers [Almeida et al., 1992], derived from the se-quence reported in the Gene Data Bank (AccessionNumber M11844).

We designed a pair of primers to amplify exon 1(9 nucleotides) as follows: ex1A 58CACAGAAGTC-CACTCATT38; ex1B 58TAGTAATAAAAGCTGGTT38.Amplification conditions for exons 1, 2, and 3 consistedof: denaturation at 94 °C for 5 min, annealing at 60 °Cfor 1 min, and elongation at 72 °C for 1 min for the firstcycle; denaturation at 94 °C for 1 min, annealing at60 °C for 2 min, and elongation at 72 °C for 1 min for 5cycles; denaturation at 94 °C for 1 min, annealing at59 °C for 2 min, and elongation at 72 °C for 2 min for 24cycles; denaturation at 94 °C for 1 min, annealing at59 °C for 2 min, and elongation at 72 °C for 7 min asthe last cycle. For exon 4, the conditions were the sameexcept for the annealing temperature, which was 61 °C(first 5 cycles) and 60 °C (last 24 cycles). PCR productswere cloned (TA-Cloning System, Invitrogen) and se-quenced (Sequenase 2.0 version Kit, Pharmacia).

Since the Thr34 mutation does not create or destroyany restriction site, we adopted the restriction-gener-ating PCR (RG-PCR) strategy to detect the mutation inthe heterozygotes. The oligonucleotide TTR34F 58AAT-GTGGCCGTGCATGTGgTCA38 (nucleotides 1670–1691) was designed and utilised with the reverse oli-

gonucleotide for exon 2 (ex2B nucleotides 1763–1744),which amplifies a 93 bp product. The lower case letterindicates a mismatched nucleotide (g instead of T),which creates a GTNAC site (MaeIII or Tsp451) only inthe PCR product derived from the mutated allele. Theamplification conditions were as follows: 94 °C for 5min, annealing at 60 °C for 1 min, and elongation at72 °C for 1 min for the first cycle; denaturation at 94 °Cfor 1 min, annealing at 59 °C for 2 min, and elongationat 72 °C for 1 min for 5 cycles; denaturation at 94 °C for1 min, annealing at 58 °C for 2 min, and elongation at72 °C for 1 min for 24 cycles; denaturation at 94 °C for1 min, annealing at 58 °C for 2 min, and elongation at72 °C for 3 min for the last cycle. The amplificationproduct was purified by PEG 6000 precipitation anddigested with MaeIII restriction enzyme following themanufacturer’s recommendations. The amplificationand the digestion products were electrophoresed on ahigh resolution agarose gel (Electran, Sigma).

RESULTSThe sequence analysis of exon 2 shows a G to C

transversion at position 1692 of the genomic sequence(Fig. 2). This mutation produces a threonine for argi-nine substitution at position 34 of the polypeptidicchain.

The Mendelian segregation of the G1692C mutationin this family was confirmed by RG-PCR followed byMaeIII digestion in two affected brothers (II-6, II-7).The young healthy daughter of II-6 (III-15) has beentested by RG-PCR together with 20 controls. More than30 normal individuals have also been analysed by ourprevious extensive sequence analysis in the TTR gene[Ferlini et al., 1992], and none carried the variant.

The individuals II-6 and II-7 (Fig. 1B, lanes c and d)show both the 93 bp uncut and the 75 bp cut products,being heterozygotes for the mutation. The female III-15(lane b) has only the uncut fragment and is thereforenot a carrier. The normal controls (lanes e, f, and g)show only the uncut fragment. Lane a shows the undi-gested amplification product; lane M is the molecularweight marker X (Boehringer).

DISCUSSIONWe report here a novel TTR missense mutation seg-

regating with hereditary amyloidosis in an Italian fam-

Fig. 1. A: Pedigree of the family carryingthe Thr34 mutation. The black box indicatesan affected patient; the shadow box repre-sents a patient reported to be affected byclinical history. The horizontal line on thesymbols is for individuals examined by ge-netic analysis. The arrow indicates the pro-positus. B: RG-PCR to detect the Thr34 mu-tation. Lane M: molecular marker X (Boe-hringer); lane a: undigested amplificationproduct (93 bp); lane b: female III-15 ampli-fication product digested with MaeIII show-ing the 93 bp uncut product only; lanes c andd: individuals II-6 and II-7 amplificationproducts digested with MaeIII showing boththe uncut (93) and cut (75) fragments; lanese, f, and g: normal control amplification prod-ucts digested with MaeIII showing only theuncut normal allele. The 18 bp fragment isnot resolved by using the agarose gel.

136 Patrosso et al.

ily. Two affected brothers carried a point mutationleading to a Thr for Arg substitution at position 34 of thepolypeptidic chain. This missense mutation does notcreate or abolish a naturally occurring restriction site,so we adopted the RG-PCR method for carrier detec-tion, confirming the mutation at the genomic level.This method allowed us to screen several control indi-viduals, which do not show this point mutation, exclud-ing the possibility that TTR Thr34 represents a normalnonpathogenic polymorphism. The biochemical analy-sis on the patient’s plasma TTR by means of hybridisoelectric focusing revealed the presence of a very un-stable mutant TTR monomer (Altland et al., unpub-lished data).

The phenotype of the individuals analysed in thepresent family is characterised by peripheral neuropa-thy and cardiomyopathy, the two most common clinicalmanifestations in TTR-HA. The clinical picture of ourpatients is indistinguishable from the phenotypic pat-tern associated with other reported TTR mutations, forexample, Ala47, Leu64, Gln89, and others [Benson,1995; Saraiva, 1995].

The clinical data from all published reports supportthe contention that most TTR mutations associatedwith hereditary amyloidosis show similar clinical con-sequences [for review see Benson and Uemichi, 1996].

Most of the amino acid substitutions cause periph-eral neuropathy, the major clinical findings in TTR am-yloidosis. Other signs are cardiomyopathy, vitreousopacities, carpal tunnel syndrome, and more rarely,other rare neurological disturbances, such as ataxia,spasticity, and dementia [Vidal et al., 1996]. All ofthese symptoms or signs are generally associated withperipheral nerve involvement and are present in vari-ous combinations. Only a few mutations such as Ile20,Lys59, Met111, and Ile122 have been reported to beassociated with an isolated organ involvement, causing

only a cardiomyopathy [Benson and Uemichi, 1996; Ja-cobson et al., 1997b]. Any amino acid substitution mayvirtually result in overlapping clinical pictures. Forthese reasons, the attempt to correlate point mutation,amino acid position, or change in the polypeptide’scharge with the clinical picture has failed, and a clearrelationship could not be established.

Another point is the clinical variability among pa-tients carrying the same point mutation. In a few cases,as for Ile50, the same substitution is responsible fordifferent organ involvement [Nishi et al., 1992; Saeki etal., 1992]. Furthermore, the absence of remarkable dif-ferences between heterozygotes and homozygotes forTTR mutations [Ferlini et al., 1996; Holmgren et al.,1992; Jacobson et al., 1997a] complicates the mattereven more.

Despite the strong evidence that amino acid substi-tutions in the TTR polypeptide chain cause hereditaryamyloidosis [Jenne et al., 1996], we conclude that otherfactors should contribute to the pathogenesis of theamyloidotic phenotype.

ACKNOWLEDGMENTS

The financial support of Telethon-Italy to A.F. (Proj-ect 1054) is gratefully acknowledged. Thanks are alsodue to the ‘‘Legato Ferrari’’ Foundation (Modena, Italy)for supporting this research in the form of a fellowship,and to the New York City Division of the AmericanHeart Association for providing support to D.R.J. Thisis publication number 12 of the Genome 2000/ITBAProject (to P.V.) founded by Cariplo. Particular thanksto Dr. Francesco Muntoni (Department of Paediatrics& Neonatal Medicine, Hammersmith Hospital, Lon-don) for the critical reading of the manuscript, and toDr. Laura Obici (Neuromuscular Unit, HammersmithHospital, London) for her technical assistance.

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