Hum Genet (1992) 89:353-356

9 Springer-Verlag1992

Transthyretin Pro 55, a variant associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurologic involvement Daniel R.Jacobson l, Dale E. McFarlin 2, lmmaculata Kane 1, and Joel N. Buxbaum 1 1Research Service, New York V. A. Medical Center, and Department of Medicine, New York University School of Medicine, New York, NewYork, USA 2Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Received February 13, 1992

Summary. Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a T--~C transversion at position 2 of codon 55, corresponding to a Leu--~ Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)primer introduced restriction analysis.

Introduction

(reviewed in Jacobson and B u x b a u m 1991). The manifestations of q-TR-amyloidosis depend largely upon the specific T T R variant present. Typically, symptoms begin from the mid 30's to 60 + years, and disease progresses over 5 - 1 0 years until death. In contrast, several m e m bers of a West Virginia kindred of Dutch and G e r m a n descent developed widespread, early-onset, rapidly progressive T r R - a m y l o i d o s i s (Fig. 1). Several reports have contained clinicopathologic data on this kindred, including immunohistochemistry, which demonstrated T r R amyloid in the tissues of two patients (Shulman and Bartter 1956; K a u f m a n 1985; Wong and McFarlin 1967; Dalakas and Engel 1981). We now present complete clinical data on this kindred and genetic studies that demonstrate a new T T R variant.

Mutations in the protein transthyretin ( T r R ) cause T T R deposition as amyloid, usually in an autosomal dominant manner, primarily in the heart, along the nerves, or both

Materials and methods

l II

l'

Ill,

[] I crg)

.

Clinicopathologic information

0 2 (aa)

[]

|

Information on all patients except II-1, which was sent to the National Institutes of Health (NIH) from the University of Virginia, was obtained during their NIH hospitalizations and on later chart review.

Genomic DNA sources, PCR, and single-stranded conformation polymorphism (SSCP) analysis

1

1

2

(191 (221 Fig. 1. Lineage of the study kindred. II, 9 Affected male, female; [2, 9 unaffected male, female; [] several unaffected siblings, male and female; ( ) age at death

Offprint requests to: D.R.Jacobson, Research Service 151, New York V.A. Medical Center, 423 East 23 Street, New York, NY 10010, USA

DNA from patients IV-1 and V-2 was isolated from Epstein-Barrvirus (EBV)-transformed peripheral blood lymphocyte lines (Jacobson et al 1984); cells from patient IV-1 were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). PCR products spanning the four TTR exons were generated using four pairs of primers, named according to Ii et al. (1992): (a) 5'(507)-22d, I2(1791)-22U; (b) I1(1515)-22D, I2(1791)-22U; (c) I2(3746)-22D, I3(4022)-22U; (d) I3(7193)-22D, 3'(7447)-24U. The products were diluted and used in 10-~tlnested PCR reactions with primers: (a) 5'(532)-22D, I1(723)-22U; (b) I1(1548)-22D, I2(1769)-22U; (c) I2(3771)-22D, I3(3996)-22U; or (d) 13(7225)22D, 3'(7418)-22U, containing 70~tM dATP, dGTP, TTP, and 35 laM dCTP, 1.8 Ixl 35S-dCTP (12.5 mCi/ml, 9.8-11.4 nmol/ml), for SSCP analysis (Orita et al. 1989). Samples were loaded onto 6% acrylamide-TBE/10% glycerol gels and subjected to electrophoresis at 800-1600 V at room temperature.

354

Sequencing of T T R exon 3 The I2(3746)-22D/I3(4022)-22U PCR fragment was gel purified, ethanol precipitated, and dissolved in 20jal of 10 m M Tris HC1/ 1 mM EDTA. Sequencing was adapted from a protocol (R. Della-

codon

55

/ \ 5 ' . . 9 GAGC~GCATGGGCTCACAACTGAGG... 3 '

5 ' 3'

CGCAeCCGAGTGTTGACTCC

. . . Ce6eGT... . . .GG~G~A...5'

Variant TTR gene sequence

3 ' 5 '

PCR-PIRA

3 '

primer

PCR product (120 bp)

BstUI

Fig. 2. Sequences of the variant gene and the P C R - P I R A primer. The variant gene contains a T--~C transition at codon 55 base 2 (boldface), which destroys an AluI (AGCT) site. The P C R - P I R A primer contains a mismatch from the target D N A (asterisk). The resulting PCR product contains a BstUI site that depends upon the presence of both the mutation and the primer-introduced substitution

Favera, personal communication) using Sequenase 2.0 kit reagents (United States Biochemical). A 6-~tl D N A sample was mixed with 3 gl 32p end-labeled I3(3996)-22U and 3.0 gl of water, heated at 95~215 l m i n , and put on ice. Sequenase 0.15 gl, 0.22gl of 100raM DTT, and 0.38~tl 5 • Sequenase reaction buffer were added to four tubes, each containing 2.5 gl of one of the four termination mixes. A 3.2@ sample of each Sequenase/termination mix was added to 2.8 gl of the primer-DNA mix at 45 ~ • 2 rain. Stop solution was added and the tubes were placed on ice. Samples were heated at 80 ~ for 3 min, placed on ice, loaded onto a 5% polyacrylamide/7M urea gel, subjected to electrophoresis, and the gel was exposed to film.

Restriction analysis and P C R - P I R A Exon 3 PCR products were AluI digested and subjected to electrophoresis on agarose gels containing ethidium bromide. PCRprimer introduced restriction analysis (PCR-PIRA) (Jacobson and Moskovits 1991; Jacobson 1992) was performed on an aliquot of the exon 3 I2(3746)-22D/I3(4022)-22U PCR product. In PCRPIRA, one primer contains a mismatch from the target DNA, such that a new restriction site is formed only in the product derived from the variant allele. Here, I2(3746)-22D and a nested 3' primer

Table 1, Clinical findings. +, Significant clinical involvement; + +, extreme involvement causing major morbidity: - , minimal or no involvement detected prior to death; NA, information not available: hct, hematocrit Patient

II-1

Ill-1

III-2

lII-5

IV-I

V-1

V-2

Sex

F

F

M

F

F

F

F

Age Disease onset Death

35 38

30 35

25 32

21 22

19 22

14 19

15 22

Heart a GI tract b

+ ++

++

++ c

+

+ +

+ ++

++ +

Weight loss

30 lb.

40 lb.

-

30 lb.

34 lb.

NA

20 lb.

++

++

+

++

++

++

+

Postural hypotension Peripheral neuropathy d

++

++

+

+

+

+

+

Ecchymoses Eye e

+ NA

+ +

++

+ -

+ +

+f

+ +f

Thyroid Liver Spleen Anemia Presenting manifestations

NA + + hct = 24 Weakness

+ + hct - 34 Irregular menses, decreased libido

+ Visionloss, goiter

+ NA NA NA Epigastricpain, vomiting

+ hct = 35 Manyg

+ Hypotension, constipation, neuropathy

+ Hypotension, nausea, epigastric pain

Other

Edema, tongue atrophy

Dehydration

Impotence, seizures

Eyelid and lacrimal edema

Vitreous veils

Corneal cloudiness

Corneal cloudiness

Amyloid + biopsies Previous references

Skin, nerve

Muscle, nerve, gum Shulman and Bartter 1956; Kaufman 1958

Vitreous

Nerve, skin

Muscle, nerve

Muscle, nerve

Muscle

Wong and McFarlin 1967

Dalakas and and Engel 1981

Dalakas and and Engel 1981

Shulman and Bartter 1956; Kaufman 1958

Shulman and None Bartter 1956; Kaufman 1958

a Constrictive cardiomyopathy: dyspnea on exertion, low-voltage EKGs, _+ chest discomfort. V-2 had left ventricular and left atrial hypertrophy on echocardiogram u Typically decreased appetite, nausea, vomiting, diarrhea, and constipation c GI symptoms were present but evidently resulted from autonomic neuropathy, not parenchymal amyloid d Included weakness, coldness, numbness, pain, decreased sensation, and decreased or absent deep tendon reflexes, beginning in

the distal lower extremities and moving centrally and later including the arms e All patients had diminished pupillary responses to light; eye involvement is considered positive only in the presence of additional signs or symptoms f Patients IV-1 and V-1 had internal ophthalmoplegia and lacrimal gland amyloidosis, impairing lacrimation and causing keratitis g Dyspnea on exertion, nausea, vomiting, anorexia, muscle weakness, dysphagia, decreased hand and foot sensation

355 GATC

containing a mismatch from the target DNA were used, leading, in the presence of the variant allele, to a product containing a BstUI site (Fig. 2).

GA, T C

Results Clinicopathologic information

G'-X_

All affected family m e m b e r s had biopsy-proven amyloidosis, multiple organ system dysfunction, and a rapid, progressive course (Table 1). Autopsies on patients III-1, III-2, I V - l , and V-2 showed heavy amyloid deposition in the heart, thyroid, blood vessels, and peripheral nerves. Gastrointestinal (GI) involvement was heavy in all patients except III-2, in w h o m involvement was limited to the G I nerves and vessels. O t h e r organs containing moderate to heavy amyloid included the liver, kidney, spleen, bladder, ovary, pituitary, uterus, pancreas, tongue, breast, muscles, dura, vocal cords, gall bladder, adrenals, thymus, lymph nodes, and skin. The retina and vitreous of patient III-2 were heavily involved, and the prostate minimally so. Deposition limited to the blood vessels and nerves but sparing p a r e n c h y m a was generally seen in the bone marrow, lungs, and brain.

G/A

D N A studies

277 - " "

SSCP analysis showed an extra band in the exon 3 P C R products from the affected kindred, indicating a variant (Fig. 3, lane 4). Exons 1, 2, and 4 from the study kindred and controls showed identical patterns. Sequencing of the exon 3 P C R product showed two bands at codon 55 position 2, one the normal sequence, and the other a T---~C change, encoding a new variant, Leu---~ Pro (Fig. 4). The T - - , C transition abolishes an A l u I site, as shown on restriction analysis (Fig. 5). Loss of this site has also been shown on Southern blotting, whereas Fnu4HI digestion of P C R products revealed a normal pattern (not shown), indicating that Southern blots showing loss of an Fnu4HI site (Jacobson et al. 1988) were incorrect. Loss of the AluI site is not specific for this mutation; any of 11

1

2

3

4

5

.../'G G

"k-c

c-./-

A Fig. 4A, B. DNA sequence around TI'R codon 55, A DNA from patient IV-l, showing both the normal (A) and variant (G) bases at codon 55 base 2 on the coding strand; transcription yields mRNA containing both the normal (CUG, Leu) and variant (CCG, Pro) codons. The rest of the sequence was normal. B To confirm the variant sequence, the abnormal allele was sequenced alone, 1 lag of genomic DNA was digested with AluI befor PCR and sequencing. Only the variant codon 55 sequence is seen. Patient V-1 DNA yielded identical results

1

2

3

1

2

.,,,-120 "'-101

179'-""

A

B

Fig. 5. A AluI digest of PCR-amplified DNA spanning TTR exon 3. Lanes I and 3 DNA from family members IV-1 and V-2; lane 2 control DNA. 40 other controls gave the pattern seen in lane 2. B BstUI digests of PCR-PIRA amplified DNA. Lane 1 patient V-2 DNA; lane 2 control DNA. Patient IV-1 showed the pattern seen in lane 1, while other controls all showed the pattern seen in lane 2

other mutations within the site would also cause its loss; therefore, P C R - P I R A provided a m o r e specific test and verified the mutant sequence (Fig. 5).

6

Discussion

Fig. 3. Exon 3 SSCP analysis. Lane 4. D N A from patient IV-l, containing the variant band arrowed. Lanes 1-3, 5 - 6 controls. Similar experiments on exons 1, 2, and 4 revealed identical patterns for the control and study samples

SSCP analysis and D N A sequencing indicated that T T R amyloidosis in this kindred was associated with a heterozygous T---~C mutation at codon 55 position 2 (Leu---~ Pro). The mutation was confirmed by AluI restriction analysis, P C R - P I R A , and sequencing of the variant allele alone. Instead of sequencing all four T T R exons, we screened each exon for a mutation by PCR/SSCP; sequencing was then limited to the relevant exon. A potential pitfall to this approach is that not all mutations are detected on all SSCP gels. To minimize the chance of missing a mutation, each P C R product was run on SSCP gels under several voltage/temperature conditions. Exons 1, 2, and 4 always showed a normal pattern for the study kindred and demonstrated the expected shifts for positive controls containing exon 2 or 4 mutations (not shown).

356 This kindred is unique for its early onset, aggressive, widespread amyloid deposition. It illustrates that the clinical picture can vary within a kindred. Patient III-2 presented with s y m p t o m s and signs caused by vitreous amyloid, whereas III-5 had decreased pupillary reflexes but no ocular s y m p t o m s and a n o r m a l slit lamp examination within a year of death. Similarly, G I tract involvem e n t ranged from heavy deposition to total p a r e n c h y m a l sparing (III-2). The heart was extensively involved in at least six of seven patients, and heart failure or arrhythmias a p p e a r e d to cause death in several. A u t o n o m i c and peripheral nervous system involvement caused postural hypotension, G I symptoms, and pupillary abnormalities in all patients. The only frequent laboratory abnormality was anemia. This family m a y exhibit the d e v e l o p m e n t and loss of a m u t a t i o n within four generations. A s s u m i n g k n o w n paternity, either patient II-1 was the founder, or one of her parents was an a s y m p t o m a t i c carrier. While carriers of some amyloidogenic T T R variants remain a s y m p t o m atic, the aggressive course suggests that in this family, gene carriers would develop disease. I n d e e d , four of seven patients developed such early, severe disease that their reproductive potential was c o m p r o m i s e d ; thus, T T R Pro 55 is the only k n o w n variant that impaired its own transmission. Anticipation is suggested, with the age of disease onset decreasing f r o m the fourth to second decade over two generations. T h e reason for m o r e aggressive disease in recent generations is u n k n o w n . Acknowledgements. This work was supported by grants from the

New York Heart Association and the Department of Veterans Affairs. We thank Dr. VictorMcKusick for information on patient III-2, and Dr. HenryF. McFarland for the B cell line from patient V-2. Drs.W. King Engel and Marinos Dalakas provided medical care at the NIH for patients V-1 and V-2.

References Dalakas MC, Engel WK (1981) Amyloid in hereditary amyloid polyneuropathy is related to prealbumin. Ann Neurol 38 : 420422 Ii S, Sobell JL, Sommer SS (1992) From molecular variant to disease: initial steps in evaluating the association of transthyretin M 119 with disease. Am J Hum Genet 50:29-41 Jacobson DR (1992) A specific test for transthyretin 122 (Val--qle) based on PCR-primer introduced restriction analysis (PCRPIRA): confirmation of the gene frequency in Blacks. Am J Hum Genet 50 : 195-198 Jacobson DR, Buxbaum JN (1991) Genetic aspects of amyloidosis. Adv Hum Genet 20 : 69-123 Jacobson DR, Moskovits T (1991) Rapid, non-radioactive screening for activating ras oncogene mutations using PCR-primer introduced restriction analysis (PCR-PIRA). PCR Methods Appl 1 : 146-148 Jacobson DR, Pras M, McFarlin DE, Buxbaum J (1988) Two new DNA-based tests for mutations causing familial amyloidotic polyneuropathy. In: Isobe T, Araki S, Uchino F, Kito S, Tsubura E (eds) Amyloid and amyloidosis. Plenum Press, New York London, PP 301-305 Jacobson S, Richert JR, Biddison WE, Satinsky A, Hartzman RJ, McFarland HF (1984) Measles virus-specific T4 + human cytotoxic T cell clones are restricted by class II HLA antigens. J Immunol 133 : 754-757 Kaufman HE (1958) Primary familial amyloidosis. Arch Ophthaltool 6:1036-1043 Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5 : 874-879 Shulman LE, Bartter FC (1956) Familial primary amyloidosis. Johns Hopkins Med Soc Bull 98 : 238-239 Wong VG, McFarlin DE (1967) Primary familial amyloidosis. Arch Ophthalmol 78 : 208-213