MUTATION NOTES 395

in TCT, which would cause a frameshift from codon 642 ( C l T a A T T + CXTGATT). This frameshift mutation would generate a stop codon immediately downstream to produce a truncated hMLHl protein.

This is the first report of a somatic mutation in DNA mismatch repair genes found in a sporadic form of endometrial cancer.

ACKNOWLEDGMENTS

Foundation, Takeda Science Foundation, and the Ichiro Kanehara Foundation. This work was supported in part by the Ministry of Education, Science and Culture of Japan, the Tokyo Biochemical Research

REFERENCES Han H-J, Maruyama M, Baba S, Park J-G, Nakamura Y (1995) Genomic structure of human mismatch repair gene, hMLHl, and its mutation analysis in patients

Liu 8 , Parsons RE, Hamilton SR, Petersen GM, Lynch HT, Watson P, Markowitz S, Willson JKV, Green J , de la Chapelle A , Kinder KW, Vogelstein B (1994)

Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction.

with hereditary nonpolyposis colorectal cancer (HNPCC). Hum Mol Genet 4:237-242.

hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindreds. Cancer Res 54: 4590-4594.

Genomics 5874-879.

A Novel Mutation (SSSSX) Causing Choroideremia; Laurent Beaufrhre, Sylvie Tuffery, Christian Hamel, Bernard Amaud, Jacques Demaille, and Mireille Claustres', Laboratoire de Biochimie GMtique, Institut de Biofogie, CNRS UPR-9008, France (L. B., S. T., J.D., M.C. ), Service dOphtafmobgie, CHU de Montpellier, France (C.H., B.A.); Fax: 33-1-6760-1 1 8 1 Communicated Thaddeus P. Dyja Received November 1, 1995; accepted March 8, 1996. 0 1996 Wiley-Liss, Inc. 'To whom reprint requestdcorrespondence should be addressed.

Choroideremia (McKusick #303100) is a rare X chromosome-linked chorioretinal degeneration. It is characterized in its later stages by severe atrophy of the outer retinal layers and choroid in the extramacular region, causing progressive nightblindness and central blindness in affected males by the third to fourth decade of life. We report a previously undescribed mutation (S558X) within exon 14 of the CHM (choroideremia) gene (van Bokhoven et al., 1994a), identified in two patients from a family originating in Southern France.

DNA extracted from peripheral blood leukocytes of nine members of the family was amplified by polymerase chain reaction (PCR) using primers described by van Bokhoven et al. (199413) and then screened for mutation through single-strand conformation analysis (SSCA), that revealed altered mobility for PCR products containing exon 14. Direct DNA sequencing after asymmetric amplification (both strands have been sequenced) revealed a C-+G transversion at nucleotide 1703, changing the serine 558 codon (TCA) to a stop ccdon (TGA). Mutation S558X creates a new restriction site for BsmAI, permitting convenient DNA-based diagnosis in the family.

This alteration, which is the most 3'-end located sequence alteration reported so far in the CHM-cDNA, is expixted to result in a product that would lack the last 96 amino acids, predicting a truncated and nonfunctional component A (REP-1) of Rat, geranylgeranyltransferase (Seabra et al., 1993). This prediction is consistent with the severe clinical phenotype observed in the affected patients.

ACKNOWLEDGMENTS

Jean-Fransois Eliaou for DNA extraction. We thank the CHM family members for their cooperation and patience. We also thank Corinne Bareil for tec,hnical assistance, and Dr.

REFERENCES Seabra MC, Brown MS, Goldstein JL (1993) Retinal degeneration in choroideremia: deficiency of Rab geranylgeranyl transferase. Science 259377-381. Van Bokhoven H, van den Hurk JAJM, Bogerd L, Philippe C, Gilgenkrantr S, de long P, Ropers HH, Cremers FPM (1994a) Cloning and characterization of

the human choroideremia gene. Hum Mol Genet 3:104-1046. Van Bokhoven H, Schwartz M, Andreasson S, van den Hurk JAJM, Bogerd L, Jay M, Ruther K, Jay 8 , Pawlowitzki IH, Sankila EM, Wright A, Ropers HH,

Rosenberg T, Cremers FPM (1994b) Mutation spectrum in the CHM gene of Danish and Swedish choroideremia patients. Hum Mol Genet 3:1047-1051.

Three Novel APC Gene Mutations in Portuguese FAP Kindreds; Brendan Marshall', Gloria Isidro, Raquel Carvalhas, Isabel Veiga, Sergio Castedo, Jose Soares, and Maria Guida Boavida, Departamento de Gm'tica Humana Instirum Nacwnal de Saiuie Dr. Ricardo Jorge, 1699 Lisboa Codex (8. M., G. I., R. C., M. G. B. ), Laboratbrio de Anatomia Patobgica, Faculdade de Medecina do Porto ( I . V. ) and Serwico de Genitica Midica, Hospital de Sdo JO&J do Porto (S.C.), 4200 Porto, Serwigo de Gastroenterofogia, Hospital Santo Antbnio do P m , 4400 Port0 (J.S.), Portugal; Fax: 351-1-759-0441 Communicated by D a d J. H. B T O C ~ Received March 11, 1996; accepted May 2, 1996. 0 1996 Wiley-Liss, Inc. 'To whom reprint requestdcorrespondence should be addressed.

Germline mutations in the APC gene on chromosome 59 are responsible for the familial adenomatous poll.posis syndrome (Grcden et al., 1991; Kinder et al., 1991). Mutations invariably lead to premature termination of translation and truncated nonfunctional proteins. During screening of Portuguese FAP kindreds, we have identified three previously unreported APC mutations using the techniques of

396 MUTATION NOTES

PCR-SSCP and the protein truncation test (PTT), which utilises in v i m transcription-translation to detect mutations that result in the introduction of a permature stop codon and thus to a truncated protein (van der Luijt et al., 1994). Exons 1-14 of the APC gene were PCR amplified using the primers described by Olschwang et al. (1993), and PCR products were separated on 6% polyacrylamide gels (acrylamide: bis 49:l) at 4°C following denaturation of PCR products. Exon 15 of the APC gene was screened for mutations using the technique of FIT with the primers described by van der Luijt et al. (1994). Exons showing abnormal SSCP patterns were reamplified and sequenced directly; samples showing truncated proteins in the PTT test were also reamplified and sequenced directly. Mutations were confirmed by their presence in all available affected individuals of the kindred as determined by direct sequencing.

In kindred “A”, a single base deletion at codon 1362 (TCC-CC) in exon 15, causing a frameshift, was identified. This results in a premature stop codon 156 nucleotides further downstream. In kindred “B’, a single base deletion at codon 592 ( lTA-+TA) in exon 14 was identified resulting in a premature stop codon 50 nucleotides down-stream. In kindred “C”, a single base C-G substitution was identified at codon 159 in exon 4 (TAC-+TACi) resulting in a T v S t o p mutation. Mutations in exon 4 have been associated with a milder form of APC known as attenuated adenomatous polyposis coli (AAPC), characterised by fewer colonic polyps, a phenotype also exhibited by affected members of kindred “C”.

REFERENCES Groden J, Thliveris A, Samowitr W, CarIson M, Gelbert L , Albertsen H, Joslyn G, Stevens J , Spirio L, Robertson M, Sargeant L, Krapcho K, Wolff E, Burt

R, Hughes JP, Warrington J, McPherson J, Wasmuth J, Le Paslier DL, Abderrahim H, Cohen D, Leppert M and White R (1991) Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66589-600.

Kinder KW, Nilbert MC, Su LK, Vogelstein 8, Bryan TM, Levy DB, Smith KJ, Preisinger AC, Hedge P, McKechnie D, Finniear R, Markham A, Groffen J. Boguski MS, Altschul SF, Horii A, Ando H, Miyoshi Y, Miki Y, Nishimo J and Nakamura Y (1991) Identification of FAP locus genes from chromosome 5q21. Science 253561-665.

Olschwang S, Laurent-Puig P, Groden 1, White R, Thomas G (1993) Germ-line mutations in the first 14 exons of the Adenomatous Polyposis Gene (AFC) gene. Am J Hum Genet 52213-279.

van der Luijt R, Meera Khan P, Vasen H, van Leeuwen C, Tops C, Roest P, den Dunnen J, Fdde R (1994) Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test. Genomics 2O:l-4.

Identification of a Novel Mutation (A268G) in Exon 8 of the HTRP Gene in a Large Family With Thyroid Hormone Resistance; Pascal Jkzkquel, Isabelle Guilhem, Jean Pierre Hespel, And& Le Treut, Jean Yves Le Gall, Hubert Allannic, and Martine Blayau’, Laboratuq of Molecular Genetics, CHU Pontckaibu, 35033 Rennes Cedex, UPR 41 CNRS, Faculty of Medicine, 35043 Rennes Cedex (P.J. A. L. T., J. Y. L. G., M. B. ), Endooinology Service, South Hospital, 35022 Rennes Cedex (1. G., 1. P. H., H. A. ), France; Fax:

Communicated by Jean-Louis M a d l Received June 16, 1995; accepted July 27, 1995. 0 1996 Wiley-Liss, Inc. ‘To whom reprint requestdcorrespondence should be addressed.

33-1 -8865-3201

Resistance to thyroid hormone (RTH) is an inherited disorder characterized by elevated concentrations of T3 and T4 and by nonsup- pressed TSH associated with manifestations suggestive of thyroid hormone deficiency or excess (Refetoff, 1993). Genetic studies have demonstrated linkage between this syndrome and the hTRp gene (Usala et al., 1988) localized on chromosome 3 and encoding a nuclear receptor for T3 (Weinberger et al., 1986).

We have analysed 22 members of a large family with RTH in which 11 patients were affected. Diagnosis was made on the basis of the following clinical and biological features: goiter, tachycardia, elevated serum concentrations of T, and T,, and normal TSH. Clinical and biochemical phenotype was similar in all affected patients. Pedigree analysis provides evidence of a dominant transmission.

DNA from the index case was first investigated. Exons 4-10 of the gene, which cover the DNA and T3 binding domains, were each amplified by PCR, and PCR products were then sequenced on both strands according to the Sanger method. PCR-mediated, site-directed mutagenesis was used to confirm the mutation identified and to screen the other family members. A heterozygous G for C substitution at nucleotide 1088, which alters codon 268 from Alanine in the wild-type to Glycine in the mutant allele (A268G), was observed in exon 8. Most of the mutations identified in the hTRp gene localize to exons 9 and 10 (Parilla et al., 1991) and only two were previously reported in exon 8 (Adams et al., 1994; Onigata et al., 1993). Eleven affected members in four generations were shown to carry the A268G mutation that was absent in 11 unaffected members. Alignment of exon 8 sequences of different species reveals conservation of alanine at position 268 in Mus Musculus, Rattus Norvegicus, Gallus Gallus, and Rana Catesbeiana. So, A268G is the very likely cause of the pathology in this family.

REFERENCES Adams M, Matthews C, Collingwood TN, Tone Y, Beck-Peccoz P, Chattetjee KK (1994) Genetic analysis of 29 kindreds with generalized and pituitary

Onigata et al. (1993) Program of the 98th meeting of the Japanese pediatric society, Yokohama, Japan. Parrilla R, Mixson A], McPherson JA, McClaskey JH, Weinrraub BD (1991) Characterization of seven novel mutations of the c-erbA p gene in unrelated

kindreds with generalized thyroid hormone resistance: Evidence in two <<hot spots>> regions of the ligand binding domain. J Clin Invest 88:2123-2130. Refetoff S, Weiss RE, Usala SJ (1993) The syndromes of resistance to thyroid hormone. Endocrine Rev 14348-399. Usala SJ, Bale AE, Gesundheit N, Weinberger C, Lash RW, Wondisford FE, McBride OW, Weintraub BD (1988) Tight linkage between the syndrome of

Weinberger C, Thomson CC, Ong ES, Lebo R, Gruol DJ, Evans RM (1986) The c-erhA gene encodes a thyroid hormone receptor. Nature (London)

resistance to thyroid hormone. Identification of thirteen novel mutations in the thyroid hormone receptor @-gene. J Clin Invest 94506-515.

generalized thyroid hormone resistance and the human c-erbA gene. Mol Endocrinol 2:1217-1220.

324:641-646.