View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Kidney Internationa4 Vol. 46 (1994), Pp.1307—1314

Mutations in the COL4A5 gene in Alport syndrome: A possible in primordial germ cells

HIT0sHI NAKAZATO, SHINZABURO HATFORI, TADASHI UsHIJwi, TosHINoBu MATSUURA, YASUSHI KoITAsHI, Tsuo TAKADA, KAzuo YosHIoKA, FuMlo ENDO, and ICHIRO MATSUDA

Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto; Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki; Department of Pediatrics, Nilgata Preftctural Yoshida Hospital, Yoshida; and Department of Pediatrics, Kinki University School of Medicine, Osaka-sayama, Japan

Mutations in the COL4ASgenein Alport syndrome: A possibleCOL4AS gene have been noted in patients with X-linked Alport mutation in primordial germ cells. Using a combination of gene amplifi- syndrome. cation with single strand conformation polymorphisms analysis and se- To initiate a search for minor mutations such as one point quencing, we examined the COL4A5genein 37 patients with Alport syndrome. In patient A8, a single base insertion was noted at codon 1,597 mutation, single base insertion or deletion in the COL4A5 gene, tyrosine in exon 49. The premature terminal signal appeared and 89 amino we carried out a combination of amplification of the exons coding acids (approximately one-third) of the non-collagenous domain were lost. for the non-collagenous (NC1) domain with single strand confor- The mutation was present in the mother, hence she is heterozygous, In patient A12, the nucleotide changed from C to Tat codon 1,679 glutamine mation polymorphisms (SSCP) [22] and sequencing of the ampli- in exon 51, which created a termination codon, and 7 amino acids at thefied DNA fragments. In 37 unrelated Japanese patients with carboxyl terminus were lost. Gene tracking using peripheral leukocytes Alport syndrome, we identified two patients with a novel mutation revealed that the parents did not carry the mutant allele, while the sister in the 3' end of the gene; one was a single base (A) insertion, was heterozygous. DNA samples from hair roots and skin fibroblasts of the resulting in a stop codon (TAA), and the other was a single base mother were normal and immunological examination of the epidermis of the mother indicated that the a5(IV) chain was normally expressed. As substitution with a nonsense mutation that was inherited from the these results suggest that somatic cells of the mother do not carry the mother. The mutation in primordial germ cells of the mother was mutant allele, the primordial germ cells possibly carry a fresh mutation in speculated in the latter case. the mother of patient A12. Methods Materials Alport syndrome [1] is an inherited progressive disease. To amplify exons 47-5 1 encoding the NC1 domain in the type The disease, which characteristically leads to end-stage renalIV a5 chain, six pairs of oligonucleotide primers were disease in affected males, is initially characterized by ,synthesized, based on the sequence of the COL4A5 gene [23] and and frequently also high-tone sensorineural deafness and eyethe flanking sequence of exon 48 (primers IVA and IVB) that we lesions. Carrier females have a variable and generally milderhave isolated and characterized [21]. The sequences of oligonu- clinical course [2]. Electron microscopy (EM) shows diffusecleotide primers were as follows: irregularly thickening and splitting of the lamina densa in theexon 51:51A:5'-GATCTGA1TGTCrFA1TFCTFATF-3' glomerular (GBM). Ultrastructural findings 51B:5'-GGACAATGAGACACFGCATCCI'A-3' and immunological evidence suggest that the principal defect inexon 50:50A:5'-TFGCGGCACA'ITfrj'CcTFGTCfl-3' the disease is due to a complete absence of, or alterations in an a 50B:5'-GGACCrGAA1T4WGCTATAAGCA-3' chain of type IV collagen, a major structural component of theexon 49:49A:5'-ATFATGTFCcTFCTCCIIIICCTF-3' GBM [3—6]. The COL4AS gene mapped to Xq22 encodes the a5 49B:5'-ATGACAAATGCAAGGAAGAGTGTA-3' chain of the type IV collagen [7, 8]. Full-length cDNA clones 48:IVA:5'-AAGGCFGGCAAGT[TI'CCflTGAAAGGCT-3' providing the complete amino acid sequence of the a5(IV) 48B:5'-CGACFAATGAATGGCTGGATGCrCF-3' collagen have been isolated and characterized [9]. Major rear- 48A:5'-AGCTGCCITCGTCGCJTFAGTACCATG-3' rangements such as large deletions [10—14] and duplication [11], IVB:5'-AGQTACCFCTGTFCFACCFAACcTAGA-3' single base mutations [9, 12, 15—18] including splicing mutationsexon 47:47A:5 '-TCTrGTATACTGATFATVFCGTGG-3' [19, 20], and single base insertion [21] in the region of the 47B:5'-CAGTAGGAAATFAGATATFGAT12A-3' Genomic DNAs were extracted from peripheral leukocytes from 37 unrelated Japanese individuals with Alport syndrome, according to the standard method [24]. Genomic DNA was Received for publication February 19, 1994 amplified in vitro by polymerase chain reaction (PCR) [25, 26]. and in revised form May 23, 1994 PCR conditions were as described elsewhere [21]. For SSCP Accepted for publication June 28, 1994 analysis, the amplified DNA was denatured at 94°C for three © 1994 by the International Society of Nephrology minutes and electrophoresed under non-denaturing conditions on

1307 1308 Nakazatoet al: Germ-line mutation in the COL4A5 gene

A B

C AS C A12 Fig. 1. PCR-SSCP-silver staining analysis. Amplified DNAs were denatured at 94°C for 3 minutes and electrophoresed on 7.5% polyacrylamide gels with 6% glycerol at 4°C at 100 volts for 10 hours. The DNA was detected after silver staining. SSCP in exon 49 (A) and exon 51(B). C, normal male control; A8, patient A8; A12, patient A12. Compared to control samples, the mobilities of the amplified DNAs of the patients were clearly exon 49 axon 51 altered, as indicated by arrowheads.

7.5% polyacrylamide gels with or without 6% glycerol at 4°C atand split into several layers (data not shown), findings compatible 100 volts for 10 hours. The DNA was detected after silver stainingwith a diagnosis of Alport syndrome. Massive (3 to 5 [27]. The amplified DNA was subcloned into the pUC18 vector,day) and hypoalbuminemia (serum albumin, 2.3 g/dl) occurred and sequenced by the dideoxy chain termination method [28]when he was 11 years old. Proteinuria (1 to 2 glday) and using 7-DEAZA Sequencing kit Ver. 2.0 (USB). For gene track-hematuria (2+ by dipstick) persist at present. Serum creatinine is ing, amplified DNAs from patients and their families were1.5 mgldl. Bilateral high tone sensorineural deafness was evident digested with restriction enzyme MseI (T/TAA), then electropho-at age four years, but the eyes remained intact. The mother and resed on 4% NuSieve GTG agarose gels. the maternal aunt have hematuria only. To determine whether somatic mosaicism was present in the Patient A12 is a 15-year-old boy with Alport syndrome without mother of patient A12, DNAs extracted from her hair roots andsensorineural deafness. Hematuria and proteinuria were detected skin fibroblasts were amplified and digested with restrictionwhen he was five years old. EM examination of glomeruli showed enzyme MseI. Ten hair roots pulled out ubiquitously were sus-findings similar to those of patient A8 (data not shown). At pended in distilled water and boiled at 94°C at 10 minutes. Nestedpresent, he has hematuria (3+) and proteinuria (± to 2+) by PCR was done using the same reaction tube. The first PCR wasdipstick, and serum creatinine is 0.8 mg/dl. High tone sensorineu- carried out with primers 50A and lB (5'-TTCTTGGAGAAAGT-ral deafness and eye lesions are absent. The parents have a normal CATTCTGGAC-3', 3'-flanking sequence of the COL4A5 geneurinalysis. He has a seven-year-old sister with hematuria (3+) and [231). Using a sample (5 .d) of the product as a template, theproteinuria (±)bydipstick, as detected at age four years. Renal second PCR was done with primers 51A and 51B. Skin fibroblastsdysfunction, and eye lesions are absent in the sister. Patient were cultured in RPMI 1640 supplemented with 10% fetal calfA12 proved to be the son of the parents, as determined by DNA serum, glutamine and penicillin/streptomycin in standard concen-fingerprints using single locus probes CMM1O1 and YNH24 (data trations, and DNA was extracted. PCR was carried out withnot shown). primers 51A and SiB. Amplified DNAs were digested with MseI and electrophoresed as described above. Results A peptide corresponding to the non-consensus amino acid We screened exons 47-5 1 coding for the NC1 domain for minor residues 1652-1664 (VDVSDMFSKPQSE) of the NC1 domain ofmutations using PCR-SSCP analysis. As shown in Figure 1, the a5(IV) chain [7] was synthesized. The synthetic peptide wasmobilities of the amplified DNAs of two patients (A8 and A12) conjugated with a carrier protein and a rat was immunized. Awere clearly different from those of control samples with regard to monoclonal antibody (H51, rat IgG2a, K-chain) was establishedexon 49 and exon 51, respectively. and it bound to peptides corresponding to the VDVSDMFSKP In patient A8, DNA sequencing of amplified products revealed region of the có(IV) NC1 peptide [29]. Indirect immunofluores-a single base (A) insertion between nucleotides A-4,993 and cence study of the a5(IV) chain of epidermal basement mem-T-4,994 at codon 1,597 tyrosine [9] in exon 49 (Fig. 2A). To brane (EBM) was carried out for the mother of patient A12. Snapexclude PCR errors, the result was confirmed by sequencing the frozen specimens of the epidermis were cut into the 2 rm-thickproducts generated by ten other independent amplifications. This sections and fixed with acetone for ten minutes then denaturedmutation led to a change from the tyrosine codon to the stop with the 6 M urea glycine HC1, pH 3.2. These preparations werecodon, TAA (Fig. 3A). The mutation resulted in synthesis of the incubated at 4°C overnight with the rat monoclonal antibody H51.truncated a5(IV) collagen chain that lacked 89 amino acids They were further incubated with fluorescein-isothiocyanate(approximately one-third) of the NC1 domain (Fig. 3A). As the (FITC) labeled goat anti-rat IgG (Capell Instruments Corp.) atmutation generated a new MseI site (restriction site, T/TAA) in 37°C for one hour, followed by Nikon fluorescence microscopy. the region of exon 49, digestion with MseI of the amplified DNAs was used for gene tracking. There was no MseI site in the Patients amplified product (175 bp) of exon 49 from the normal control Patient A8 is a 12-year-old boy with Alport syndrome and(Fig. 4). When the amplified DNA from the patient was digested sensorineural deafness. Gross hematuria occurred when he was 14with MseI, a 175 bp fragment was absent and new fragments (130 months old and proteinuria was evident when he was four yearsand 46 bp) appeared (Fig. 4). The sample from the mother of old. EM examination of glomeruli obtained at renal biopsy at thepatient A8 contained fragments of 175 bp, 130 bp and 46 bp by latter age revealed that the lamina densa of GBM had widenedMseI digestion, indicating that she was heterozygous with wild Nakazato et al: Genii-line mutation in the COL4A5 gene 1309

normal A8

1A 4993 T 4994 T

normal Al 2 GATCGATC

Fig.2. Sequencing of PCR amplified DNAs. A Partial nucleotide sequences of amplified — T 5238 DNAs with primers 49A and 49B (A), and T primers 51A and SiB (B). Left, normal male G control; right, patients AS (A) and A12 (B). A. The mutation in patient AS proved to be a single base insertion (indicated by arrowheads) between nucleotides A-4993 and T-4994 [11]. B. In patient A12, nucleotide 5,238 was changed from C to T indicated by arrowheads. type and mutant alleles. DNA samples from other members of theand fibroblasts showed the normal fragment (138 bp) only (Fig. 6). family could not be obtained. These results suggest that the mother of patient A12 is not In patient A12, nucleotide 5,238 was changed from C to T,heterozygous, at least with regard to peripheral leukocytes, hair converting codon 1,679 glutamine in exon 51 to stop codon, TALkroots and cultured skin fibroblasts. Immunofluoreseence study (Figs. 2B, 3B). The result was confirmed by the procedurewith the anti-aS(IV) antiserum gave linear stainings for the described above. This mutation resulted in synthesis of a trun-epidermal basement membrane of the mother, findings similar to cated protein lacking seven amino acid residues at the earboxylthat of a normal male control (Fig. 7). terminus (Fig. 3B). The amplified product (192 bp) in exon 51 from the normal control was digested with MseI, producing two Discussion fragments (138 and 54 bp) (Fig. 5). In the patient, a fragment of We examined the 3'-end of the COMAS gene in 37 Japanese 138 bp was absent and two new fragments (88 and 50 bp), inpatients with Alport syndrome, using PCR-SSCP-silver staining addition to an original 54 bp fragment, were observed, because ananalysis, and we identified five mutations; one patient with a single additional MseI site had formed with the mutation in exon 51 (Fig.base insertion [21], two with a splicing mutation (in preparation), 5). The sample from the sister of the patient contained fragmentsand two patients in this study. The mutations heretofore reported of 138 bp, 88 bp, 54 bp and 50 bp, indicating that she was[9—20] differed from those found in our patients and were heterozygous with the mutant and the wild type alleles. The fatherubiquitously in the COMM gene from triple-helical to NC1 and the mother carried only the wild type alleles. domains. The detection rate of mutations by our methods, which We investigated additional DNA samples obtained from hairwas limited to identification of mutations located at the NC1 roots and cultured skin fibroblasts from the mother of patientdomain, was 13% (5 of 37). In the remaining 32 patients, no A12. MseI digestion revealed that amplified DNAs from hair rootsabnormal bands were detected in five exons 47-5 1 covering the 1310 Nakazatoet afr Germ-line mutation in the COL4A5 gene

Amino acid 1597 C W D S L W I G Y S Normal GGA TCC CATTCT CTC TGG APr GGT TAT TCC I, Patient A8 GGA TGG CAT TCT CTG TGG AlT GGT TAA TFC G W D S L W IC *

4 Helical '4 Nd domain

Fig. 3.The amino acid sequence and deduced Amino acid 1679 1685 protein of the a5(Ik9 collagen chain of patients. * Top. Amino acid numbers start at the ItC Q V£ M K R T translation initiator methionine [11]. Normal CCATGTCAAGTGTGCATC AAG AGG ACA TAA Nucleotide and deduced amino acid sequences in normal control and patients are 1' shown by inner and outer lines, respectively. CGA TGT TAA GTGTGC ATG AAG AGG ACA TAA The mutations are indicated by arrowheads, Patient A12 and stop codons by asterisks. Bottom. Illustration of deduced protein of the a5(IV) collagen chain of patients. A. A single base insertion in patient A8 led to change from 1597 tyrosine codon into stop codon, TAA. The deduced protein would lack about one- third of the NC1 domain shown by the gray box. B. A single base mutation in patient A12 led to a change from 1679 glutamine codon into stop codon, TAA. The deduced protein would lack 7 amino acids (1679.1685) at its carboxyl terminus shown by the gray box. iØ— Helical NC1 domain

NC1 domain. They may have a mutation in other exons of thethe carboxyl terminus were absent, as a result of a nonsense COL4A5 gene encoding the triple helical domain, or the COL4A6mutation. gene mapped to Xq22 encoding the newly found a6(IV) chain The NC1 domain seems to be crucial for the correct alignment [30]. Further studies on the remaining regions are needed. of three a chains prior to their folding into the triple-helical In patient A8, the mutation in exon 49 resulted in the absenceconformation in a manner similar to that in [31]; of approximately one-third of the NC1 domain of a5(IV) chain.it is essential for the head-to-head cross-linking between the We found a single base insertion in exon 48 in another patientcarboxyl terminals of two triple-helical molecules during forma- (OYM) [21]. Both mutations created a terminal codon and bothtion of the type IV collagen network [32, 33]. These functions patients had similar clinical findings of the X-linked juvenile formrequire the correct conformation of the NC1 domain that is of Alport syndrome with high-tone sensorineural deafness,maintained by disulfide bonds. The truncated NC1 domain found though the extent of the absence of the NC1 domain in patientin patient A8 would mean that the structure of type IV collagen OYM was greater than that in patient A8 (2/3 vs. 1/3) [21]. In patientwould not develop normally. In patient A12, the lack of the last A12, seven amino acid residues 1,679 to 1,685 (QVCMKRT) atcysteine residue of the NC1 domain, which contains 12 cysteine Nakazato et a!: Germ-line mutation in the COL4A5 gene 1311

A A

2 2

II 1 II I 2

B * B Exon49 I Normal 175 Exon5l

A8 Normal 138 I 130 46 54 Al 2 88 50 54 C

M C 12 lii bp

175 C 130 M Ii 12 Ill 112 C M bp 4 438

46 88 454 (50)

Fig. 4. Pedigree of patient A8 and gene tracking by restriction enzyme MseI Fig. 5. Pedigree ofpatientAl2 and gene tracking by restriction enzyme MseI digestion of amplified DNA. A. Pedigree of patient A8. Obligate female digestion of amplified DNA. A. Pedigree of patient A12. Unaffected female carrier is shown as an open circle with a dot. Unaffected male and affected is shown as an open circle. See Fig. 4A. B. Region of amplified DNA with male are shown as an open box and a closed box, respectively. The patient primers 51A and 51B. See Fig. 4B. C. Digestion of amplified DNA with is indicated by an arrowhead. B. Region of amplified DNA with primersMseI and electrophoresis on a 4% NuSieve gel. Ii, father; 12, mother; 111, 49A and 49B. The mutation is indicated by an asterisk. MseI restriction patient; 112, sister; C, normal male control; M, 1kb ladder marker. The site is indicated by a vertical line. C. Digestion of amplified DNA with patient is missing the 138 bp fragment and has variant fragments of 88 bp MseI and electrophoresis on a 4% NuSieve gel. C, normal male control; 12, and 50 bp. The sister is heterozygous with the normal and the variant mother; Hi, patient; M, 1kb ladder marker. The patient is missing the 175 fragments. The father and the mother have only a normal fragment. bp fragment and has the variant 130 bp and 46 bp fragments. His mother is heterozygous with the normal and the variant fragments.

residues, would abolish one of the intra-chain or inter-chaincarried only wild type alleles. Thus, further detailed studies on the disulfide bonds. Accordingly, although protein examination wasmother are needed, since the disease is inherited in an X-linked not done, it is likely that mutations are causative for the disease.fashion. First, we examined paternity and maternity of the chil- Concerning the heredity of the disease in the patient A8 family,dren, using DNA fingerprints. We also did a gene analysis of the mutation was inherited from the maternal side. In the familysomatic cells of the mother in addition to peripheral leukocytes, of patient A12, the parents are healthy with no urine abnormality,since carriers of Lesch-Nyhan syndrome with X-linked inherited yet the patient and his sister have had hematuria and proteinuria.disease often have the wild type allele alone in peripheral Gene tracking by use of MseI digestion revealed that the parentsleukocytes [34]. MseI digestion showed that both hair roots 1312 Nakazatoet a!: Germ-line mutation in the COL4A5 gene

1 2 3 4 5 6 M bp

192

-.4138

88

Fig. 6. MseI digestion of amplified DNAs from hair roots and cultured skin fibroblasts of patient A12's mother. Ten hair roots pulled out ubiquitously were suspended in distilled water and boiled at 94°C at 10 minutes. Nested PCR was done. First PCR was carried out with primers 50A and lB.Using the product as a template, a second PCR was done with primers 51A and 51B. The DNA extracted from cultured skin flbroblasts was amplified with primers 51A and 51B. Amplified DNAs were digested with restriction enzyme MseI (lanes 2, 4 and 6) or without the enzyme (lanes 1, 3 and 5),and electrophoresed on a 4% NuSieve gel. Leukocytes in a normal male control (lanes 1 and 2), hair roots (lanes 3 and 4) and cultured skinflbroblasts (lanes 5 and 6) in the mother of patient A12; M, 1kb ladder marker. A fragment of 54 bp ran off the gel. The mother hadthe normal fragment (138 bp) in hair roots and skin flbroblasts but not the variant one (88 bp).

Fig. 7. Indirect immunofluorescence study of epidermal basement membrane (EBM) with anti-o5(IV) antibody H51 in the patient A12's mother and a normal male control. The 2 inn-thick cryosections were incubated at 4°C overnight with the rat monoclonal antibody against the peptide of NC1 domain of a5(IV) chain. The sections coated with primary antibody were incubated with FITC-labeled goat anti-rat IgG at 37°C for one hour, followed by Nikon fluorescence microscopy. The EBMs are indicated by arrowheads. A. patient A12's mother, (X400). B. normal male control, (X354). The EBMs of patient A12's mother and a normal male control are strongly stained by anti-a5(IV) antibodies.

(ectoderm) and skin fibroblasts (mesoderm) from the mothermarkers indicated that the mutation was transmitted from the carried the wild type allele of the COL4A5 gene alone. Anunaffected father [37]. A most plausible explanation is that the indirect immunofluorescence pattern of EBM was similar to thatmutation must have occurred during mitosis in early germ-line seen in normal controls. In male patients with Alport syndrome,proliferation. The studies presented here showed that the muta- neither GBM nor EBM reacted with H51 [291. In heterozygoustion in family A12 occurred at some stage after dividing primor- females; there was a discontinuous or mosaic pattern in thedial germ cells from somatic cells during the embryonic develop- immunofluorescent staining of the GBM and EBM with FNS1ment of the mother, since she carried the wild type allele alone at [35] and H51 [29]. Based on these findings, the mother does notleast in three somatic tissues and if studies on the epidermis seem to be a heterozygous female with Alport syndrome. Arevealed normal findings. If the mutation occurred early in similar situation was noted in Duchenne muscular dystrophy,embryogenesis, the primordial germ cells would be a homologous another X-linked inherited disease [36, 37]. Bakker et al describedmutation, while if later, they would be mosaicism. The recurrence two families in which deletion of the dystrophin gene was trans-risk would be related to the extent of the subsequent germ-line mitted to more than one offspring by women with no evidence formosaicism. Murray, Giles and Lillicrap reported that in a family the mutation in their own somatic (white blood) cells [361. Darraswith type JIB von Willebrand disease, analysis of the sperm from and Francke reported that deletion of the dystrophin gene wasa phenotypically normal grandfather showed that approximately present in two of five daughters of a woman who herself did not5% of the germ-line contained the mutant sequence [38]. In the have the mutation, and restriction fragment length polymorphismfamily A12, informed consent for the ovary biopsy could not be Naknzato et aL Germ-line mutation in the COL4A5 gene 1313 obtained from the mother. Ovary or ovum examinations may be12. SMEETS HiM, MELENHORST JJ, LEMMINK HH, SCHRODER CH, NELEN revealing. It is possible that a fresh mutation of the COL4A5gene MR, Zi-tou J, Hosriice SL, TRYGGVASON K, ROPERS HIT, JANSWEI- in primordial germ cells (homologous mutation or mosaicism) in JER MCE, MONNENS LAH, BRUNNER HG, v OosT BA: Different mutations in the COL4A5 collagen gene in two patients with different the mother results in the hemizygote (patient A12) and the features of Alport syndrome. Kidney mt 42:83—88, 1992 heterozygote (a sister of the patient) of Alport syndrome. The 13. RENmiti A, SERI M, MYERS JC, PIIIWANIEMI T, SESSA A, RIZZONI G, mutation will be passed down to their offspring. MARCHI MD: Alport syndrome caused by a 5' deletion within the In subjects with Alport syndrome, familiar gene tracking was COL4A5 gene. Hum Genet 89:120—121, 1992 carried out in 17 families, including present cases, and de novo 14. NETZER KO, RENDERS L, ZHOU J, PULLIG 0, TRYGGVASON K, WEBER M: Deletions of the COL4A5 gene in patients with Alport syndrome. mutation was found three families (3 of 17, 17%) [14, 18]. The Kidney Int 42:1336—1344, 1992 figure is close to that reported (18%) by Shaw and Kallen [39]. 15. ZHOU J, BARKER DF, HOSTJKKA SL, GREGORY MC, AnoN CI. We conclude that gene analysis including familiar gene tracking TRYGGVASON K: Single base mutation in a5 (IV) collagen chain gene is important, in addition to pathophysiological studies of the converting a conserved cysteine to serine in Alport syndrome. Genom- ics 9:10—18, 1991 propositus. 16. ZHOU J, HERTZ JM, TRYGGVASON K: Mutation in the a5(IV) collagen chain in juvenile-onset Alport syndrome without or ocular Acknowledgments lesions: Detection by denaturing gradient gel electrophoresis of a PCR product. Am J Hum Genet 50:1291—1300, 1992 We are grateful to the pediatricians at 17 hospitals in Japan for 17. KNEBELMANN B, DESCHENES G, GROS F, HoRs MC, GRUrcruu JP, providing blood samples from patients with Alport syndrome, to A. Koide, TRYGGVASON K, GUBLER MC, ANTIGNAC C: Substitution of arginine K. Uchikawa, Drs. T. Kawano and R. Hoshide for technical assistance, to for glycine 325 in the collagen a5(IV) chain associated with X-linked Dr. Y. Ohsuga (Department of Dermatology, St. Marianna University Alport syndrome: Characterization of the mutation by direct sequenc- School of Medicine) for skin biopsy, to Drs. S. Tsunenari and C. Toki ing of PCR-amplified lymphoblast eDNA fragments. Am JHum Genet (Department of Forensic Medicine, Kumamoto University School of 51:135—142, 1992 Medicine) for DNA fingerprints, and to M, Ohara for critical comments. 18. RENTER! A, SERI M, MYERS IC, PIHLAJANIEMI T, MASSELLA L, RIZZONI G, M.&itcrn M: De novo mutation in the COL4A5 gene Reprint requests to Shinzaburo Hattori, M.D., Department of Pediatrics, converting glycine 325 to glutansic acid in Alport syndrome. Hum Mol Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Genet 1:127—129, 1992 Japan. 19. NETTER KO, PULLIG 0, FRET U, Zi-rou J, TRYGGVASON K, WEBER M: COMAS splice site mutation and a5(IV) collagen mRNA in Alport References syndrome. Kidney mt 43:486—492, 1993 20. NOMURA S, OSAWA G, SM T, H.kiw'io T, HAIW.io K: A splicing 1. ALPORT AC: Hereditary familial congenital haemorrhagic nephritis. mutation in the a5(IV) collagen gene of a family with Alport's Brit Med J (Clin Res) 1:504—506, 1927 syndrome. Kidney mt 43:1116—1124, 1993 2. FLINTER FA, CAMERONJS,CHANrLER C, HOUSTON I, Bointow M: 21. NAKAZATO H, HATrORI S, MATSUURA T, KOITABASHI Y, ENDO F, The genetics of "classic" Alport's syndrome. Lancet 2:1005—1007,1988 MATSUDA I: Identification of a single base insertion in the COMAS 3. HINGLAS N, GRUNFELD JP, Bois E: Characteristic ultrastructural gene in Alport syndrome. Kidney mt 44:1091—1096, 1993 lesion of the glomerular basement membrane in progressive heredi- 22. ORITA M, IWAHANA H, KANAZAWA H, HAYASHI K, SEKIYA T: tary nephritis (Alport syndrome). Lab Invest 27:473—487, 1972 Detection of polymorphisms of human DNA by gel electrophoresis as 4. Yosiiw N, CAMERON AH, Wi-nm HR: The glomerular basal single-strand conformation polymorphisms. Proc Nat! Acad Sci USA lamina in hereditary nephritis. J Pathol 135:199—209, 1981 86:2766—2770, 1989 5. McCoi RH, JOHNsoN K, STor'm Wi, WILSON CB: Absence of 23. ZHOU I, HosTixxA SL, CHOW LT, TRYGGVASON K: Characterization nephritogenic antigen(s) in some patients with hereditary nephritis. of the 3' half of the human type IV collagen aS gene that is affected Kidney mt 21:642—652, 1982 in the Alport syndrome. Genomics 9:1—9, 1991 6. KLEPPEL MM, KsIuAr C, SANrI PA, WIESLANDER J, MICHAEL AF: 24. SAMBROOK I, FRITSCH EF, MANIATIS T: Molecular Cloning. A Labo- Distribution of familial nephritis antigen in normal tissue and renal ratory Manual (2nd ed). New York, Cold Spring Harbor Laboratory basement membranes of patients with homozygous and heterozygous Press, 1989 Alport familial nephritis. Relationship of familial nephritis and Good- 25. SAJICI RK, Sctwu S, FALOONA F, Muws KB, Hoit GT, ERLICH HA, pasture antigens to novel collagen chains and type IV collagen. Lab ARNHEIM N: Enzymatic amplification of 13-globin genomic sequences Invest 61:278—289, 1989 and restriction site analysis for diagnosis of sickle cell anemia. Science 7. HOSTIKKA SL, EDDY RL, BYERS MG, HOYffrYA M, SHOWS TB, 230:1350—1354, 1985 TRYGGVASON K: Identification of a distinct type IV collagen a chain 26. SAIIZI RK, GELFAND DH, STOFFEL S, SCHARF Si, HIGUCHI R, HORN with restricted kidney distribution and assignment of the gene to the GT, MULLTS KB, ERLICH HA: Primer-directed enzymatic amplifica- locus on X-linked Alport syndrome. Proc NatlAcad Sci USA 87:1606— tion of DNA with a thermostable DNA polymerase. Science 239:487— 1610, 1990 491, 1988 8. PrnIAJmMI T, POHJOLATNEN ER, MYERS JC: Complete primary 27, HOSHIDE R, MATSUURA T, KOMAKI S, Kouce E, URN0 I, MATSUDA I: structure of the triple-helical region and the carboxyl-terniinal domain Specificity of PCR-SSCP for detection of the mutant ornithine of a new type IV collagen chain, a5(IV). J Biol Chem 265:13758— transcarbamylase (OTC) gene in patients with OTC deficiency.Jlnher 13766, 1990 Metab Dis 16:857—862, 1993 9. ZHOU J, HERTZ JM, LEINONEN A, TRYGGVASON K. Complete amino 28. SANGER F, NICKLEN S, CouLSoN AR: DNA sequencing with chain acid sequence of the human a5(IV) collagen chain and identification terminating inhibitors. Proc NatlAcad Sci USA 74:5463—5467, 1977 of a single-base mutation in exon 23 converting glycine 521 in the 29. Yosi-nOKA K, HINO S, TAKEMURA T, Mio S, WIESLANDER I, TAKE- collagenous domain to cysteine in Alport syndrome patient. J Biol ROSIn Y, MAKINO H, KAGAWA M, SAIx) Y, KASHTAN CE: 1rpe IV Chem 267:12475—12481, 1992 collagen aS chain: Normal distribution and abnormalities in X-linked 10. BARKER DF, Hosmn SL, ZHOU J, Ciow LT, OLIPI-IANT AR, Alport syndrome revealed by monoclonal antibody. Am J Pathol GERKEN SC, GREGORY MC, SKOLNICK MH, ATEIN CL, TRYGGVASON 144:986—996, 1994 K: Identification of mutations in the COI.4A5 collagen gene in Alport 30. ZHOU I, MOcHIzUKI T, SMEETS H, ANTIGNAC C, L4.uluLA P, PAePE A, syndrome. Science 248:1224—1227, 1990 TRYGOVASON K, REEDERS ST: Deletion of the paired a5(IV) and 11. Bom E, VETRIE D, FLINTER F, BUCKLE B, PINLAJANIEMI T, H.- a6(IV) collagen genes in inherited smooth muscle tumors. Science MALAINEN ER, MYERS JC, BoaRow M, HARRIS A: Major rearrange- 261:1167—1169, 1993 ments in the a5(IV) collagen gene in three patients with Alport31. DOEGE KJ, FESSLER JH: Folding of carboxyl domain and assembly of syndrome. Genomics 11:1125—1132, 1991 procollagen I. J Biol Chem 261:8924—8935, 1986 1314 Nakazato et al: Germ-line mutationin the COL4A5 gene

32, WEBER S, ENGEL J, WIEDEMANN H, GvILu RW, TIsn'L R: membranes of kindreds with Alport-type familial nephritis. J Clin Subunit structure and assembly of the globular domain of basement- Invest 78:1035—1044, 1986 membrane collagen type IV. Eur J Biochem 139:401—410, 1984 36. BiocER E, v BROECKHOVEN CH, BONTEN EJ, VAN DE VOOREN MJ, 33. SIEBOLD B, DEUTZMAN R, KUHN K: The arrangement of intra- and VEENEMA H, VAN HUL W, AN OMMEN GJB, VANDENBERGHE A, intermolecular disulfide bonds in the carboxyterminal, non-collage- PEARSON PL: Germline mosaicism and Duchenne muscular dystrophy nous aggregation and cross-linking domain of basement membrane mutations. Nature329:554—556, 1987 type IV collagen. Eur J Biochem 176:617—624, 1988 37. Dius BT, FEANcIce U: A partial deletion of the muscular dystrophy 34. McKuiw RO, HOWELL A, ANDREWS TM, WATTS RWE, Ainrr gene transmitted twice by an unaffected male. Nature 329:556—558, CF: Observations on the growth in vitro of myeloid progenitor 1987 cells and fibroblasts from hemizygotes and heterozygotes for "com- 38. MURRAY EW, GILES AR, LILLICRAP D: Germ-line mosaicism for a plete" and "partial" hypoxanthine-guanine phosphoribosyl-trans- valine-to-methionine substitution at residue 553 in the glycoprotein ferase (HGPRT) deficiency, and their relevance to the pathogenesis lb-binding domain of von Willebrand factor, causing type TIB von of brain damage in the Lesch-Nyhan syndrome. J Neurol Sci 22:183— Willebrand disease. Am J Hum Genet 50:199—207, 1992 195, 1974 39. Sii&w RF, KALLEN RI: Population genetics of Alport's syndrome: 35. KASHTAN C, Fisn AT, KLEPPEL M, YosmoEA K, MICHAEL AF: Hypothesis of abnormal segregation and necessary existence of mu- Nephritogenic antigen determinants in epidernial and renal basement tation. (abstract) Nephron 16:427, 1976