Sequences in the hypervariable region 1 of hepatitis C virus show only minimal variability in the presence of antibodies against hypervariable region 1 during acute infection in chimpanzees.

We analyzed sequences of hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in six chimpanzees, experimentally infected with a single HCV inoculum, to clarify the correlation between HVR1 mutation and antibodies to HVR1. Two chimpanzees had been immunized with synthetic HVR1 peptides before HCV inoculation. All six animals became infected with HCV but cleared the infection within the acute phase. The major HVR1 sequences in longitudinal sera were unchanged in animals both with and without anti-HVR1 antibodies. Additionally, sequences of HVR1 variants in each chimpanzee converged after 11 to 19 weeks. The data show that anti-HVR1 antibodies are unlikely to drive variation in HVR1.

Abnormal nucleotide repeat sequence in the TGF-betaRII gene in hepatocellular carcinoma and in uninvolved liver tissue.

Replication error (RER)-related genetic alterations are associated with a subset of hepatocellular carcinomas (HCCs) with multiple primary cancers. This study investigated whether mutations in the nucleotide repeats of three putative target genes of RER are associated with hepatocarcinogenesis. The genes examined were those encoding transforming growth factor beta type II receptor (TGF-betaRII), BCL-2-associated X protein (BAX), and insulin-like growth factor II receptor (IGF-IIR). Tumour and non-tumour hepatic tissues were examined in 48 HCC patients, 34 with solitary HCC and 14 who had double cancer with gastric cancer. Four double-cancer cases showed an abnormal signal in the single nucleotide repeat (A)10 of the TGF-betaRII gene. These four were among the six RER-positive cases in this series. The genotypes of the poly A tract of the TGF-betaRII gene in the liver tumour tissue of the four cases with an abnormal signal were (A)9/10, (A)9/10, (A)9/10, and (A)9/9. Five uninvolved liver tissue specimens from these four patients showed (A)9/10 and (A)9/9, (A)9/10, (A)10/10 and (A)9/9, respectively. The genotype in the stomach cancer specimens of these four patients was (A)10/10, indicating no germline mutation of the TGF-betaRII gene. There were no mutations in the nucleotide repeats of the BAX and IGF-IIR genes in any of the liver tissue specimens. Abnormality of the nucleotide repeat in the TGF-betaRII gene occurred in the uninvolved liver tissue as well as the HCC tissue in some HCC patients. Such genetic instability may be gene-specific and tissue-specific in carcinogenesis.

Distinct relationship between polypoid growth type and sporadic colorectal carcinomas with microsatellite instability.

BACKGROUND/AIMS: This study was carried out to clarify whether colorectal carcinomas with MSI (microsatellite instability) is correlated with growth types of invasive carcinomas. METHODOLOGY: Samples of tumor tissue and adjacent normal mucosa were obtained from 45 patients with sporadic advanced colorectal cancer. The MSI was assessed by the mobility shift assay of microsatellite and VNTR (variable numbers of tandem repeat) alleles using 12 markers. Tumors with four or more positive loci were determined to be MSI positive. The polyadenine tract (A)10 of the third exon in TGF beta RII was also assessed by mobility shift assay of DNA fragments amplified with primers. Histological examination was performed to divide all tumors into polypoid growth carcinoma and nonpolypoid growth carcinoma, according to Shimoda et al.'s classification. RESULTS: Ten of 11 cases with MSI had a 1-base pair deletion in a polyadenine tract in the TGF beta RII gene. Fifteen cases showed polypoid growth and 30 cases indicated nonpolypoid growth. There were 9 polypoid growth cases and 2 nonpolypoid growth cases with MSI, while there were 6 polypoid growth cases and 28 nonpolypoid growth cases without MSI. Colorectal cancer cases with MSI had a significantly higher incidence of cases with polypoid growth (9/11) compared to those without MSI (6/34) (P = 0.0004). CONCLUSIONS: Sporadic colorectal carcinomas with MSI tend to show a polypoid growth type. We think that there are two types including "adenoma-carcinoma sequence" type and "RER" type in colorectal carcinomas that show adenoma-carcinoma progression.

Prevention of hepatitis C virus infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1.

We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.

Phylogenetic investigation for the risk of hepatitis C virus transmission to surgical and dental patients.

Blood loss during treatment carries a potential risk for the transmission of blood-borne pathogens in hospital patients. To determine whether nosocomial transmission of hepatitis C virus (HCV) occurs in surgical wards and dental hospitals, we tested anti-HCV antibodies and HCV RNA in sera from these patients and analysed the hypervariable region 1 (HVR1) sequence of HCV phylogenetically in the HCV RNA-positive patients. Five of 83 patients from a surgical ward were positive for HCV RNA, and six patients from one dental hospital and nine patients from a second were found to be positive for HCV RNA during the examination period. The HVR1 sequences were amplified from these patients' serum, and after subcloning, multiple clones of the HVR1 sequence from each patient were determined. The phylogenetic analysis of these sequences showed that HVR1 species from each patient could be classified into one to three genetic clusters of HVR1 quasi-species and that these clusters were independent of each other among patients. Thus, there was no evidence of HCV transmission in our study, and unrecognized transmission of HCV may be a rare event in surgical and dental patients at university hospitals.

Expression of peroxisome proliferator-activated receptor subtypes in human atherosclerosis.

To clarify the involvement of peroxisome proliferator-activated receptors (PPARs) in atherosclerotic plaque formation, we investigated the expression patterns of mRNA and protein of PPARalpha and PPARgamma in human aorta. Atheromatous plaque, fatty streak, and diffuse intimal thickening (DIT) were separated macroscopically, and each sample was divided into halves. Half of them were used for analysis of mRNA expression with reverse transcription-polymerase chain reaction and the others were used for histologic analysis. Both PPARalpha and PPARgamma mRNA were detected in all atheromatous plaques, all fatty streaks, and in some DIT. However, expressions of PPARalpha and PPARgamma were obviously less frequently found in DIT than in atheromatous plaques, and the intensity of these expressions was stronger in the atheromatous plaques than in the DIT. Compared with PPARalpha, PPARgamma mRNA was expressed more frequently in atheromatous plaques. In atheromatous plaques, PPARgamma mRNA was expressed independently, whereas PPARalpha mRNA was coexpressed with PPARgamma. PPARgamma protein was obviously found in the nuclei of endothelial cells, macrophages, mononuclear cells, and smooth muscle cells in the aortic intima. These results suggest that expressions of PPARalpha and PPARgamma in human aortic wall are involved in atherogenesis from the early stages.

Phylogenetic evidence, by multiple clone analysis of hypervariable region 1, for the transmission of hepatitis C virus to chronic haemodialysis patients.

We analysed hepatitis C virus (HCV) sequences to determine whether nosocomial transmission of HCV occurred in a haemodialysis unit. Twenty patients positive for serum HCV RNA were investigated. All were undergoing haemodialysis therapy in the same room. The hypervariable region 1 (HVR1) sequence of HCV was amplified and multiple clones sequenced. Phylogenetic analysis of these sequences revealed five genetic clusters consisting of HCV isolates from 11 of the 20 patients. In addition to two genetic clusters of HCV isolates from the four currently seroconverting patients and another patient who had been persistently infected, we identified three other phylogenetic relationships in HCV isolates from six patients. The patients grouped into the same cluster received haemodialysis individually on the same shift and/or side-by-side. Phylogenetic analysis of HCV HVR1 sequences corroborated the patient-to-patient HCV transmission suggested by an epidemiological study and that unrecognized transmission of HCV occurs in the dialysis room. Our multiple clone analysis of HCV isolates provides detailed information on nosocomial transmission of HCV. Transmission occurs more frequently when treatment is performed at the same time than in consoles located close to each other.

Occasional infection of hepatitis C virus occurring in haemodialysis units identified by serial monitoring of the virus infection.

To examine whether hepatitis C virus (HCV) infection still occurs in haemodialysis units even after a decrease in the number of blood transfusions and in those contaminated with HCV, we tested anti-HCV antibodies and HCV RNA in 142 patients from Japanese haemodialysis units, and examined the serial prevalence of anti-HCV antibodies in 86 patients from 1992 to 1997. A high prevalence of HCV infection was observed: 34 (23.9%) and 38 (26.8%) of the 142 patients were positive for serum anti-HCV antibodies and HCV RNA, respectively. These positive rates were related to the duration of haemodialysis. We found that five patients treated in the same unit seroconverted from 1993 to 1995. Four of the five patients had been treated at the same shift and were affected at the same time. Phylogenetic analysis of the hypervariable region 1 (HVR1) sequence of HCV from serum of these patients showed that three of the four patients' sequences were phylogenetically clustered and that two of the three were closely related. Thus, an occasional transmission of HCV occurred in the haemodialysis unit. The universal precautions by staff such as carefully changing gloves may be important in inhibiting spread of HCV because no instances of infection have been seen since the instigation of educational measures for unit staff.

Expression of muscarinic receptor genes in the human coronary artery.

The authors investigated the role of muscarinic receptors in functional control of coronary arteries affected by intimal thickening due to arteriosclerosis. They first examined the genetic subtypes of muscarinic receptors expressed in human coronary arteries. Twelve samples of human coronary artery, obtained by autopsy from eight subjects, were examined for the expression of four genetic subtypes of muscarinic receptor, m1 to m4, by reverse transcription and polymerase chain reaction (RT-PCR). Two subtypes, m2 and m3, were found to be expressed in the coronary artery. The m2 gene was expressed in seven of the 12 vessels, and m3 in eight of the 12. Expression of both m2 and m3 genes was observed in five of the 12 vessels. Neither the m1 nor m4 was expressed in these samples. These results indicate that the m2 and m3 genes are mainly expressed in the coronary arteries and suggest that these patterns of expression are differentially controlled to induce the diversity of contraction/relaxation reactions induced in the coronary arteries by acetylcholine.

Monoclonal antibodies to the hypervariable region 1 of hepatitis C virus capture virus and inhibit virus adsorption to susceptible cells in vitro.

To analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner.

Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees.

A chimpanzee was immunized with two recombinant envelope glycoproteins E1 and E2 of hepatitis C virus (HCV), strain HCV-N2, and the hypervariable region 1 (HVR1) peptides of a different isolate, HCV-#6, then received an intravenous inoculation of 10 chimpanzee infectious doses of HCV-#6. With high humoral immune response against E1 and E2 but a low response against HVR1, the vaccinee became infected with the HCV. However, after increasing the titer of anti-HVR1 against HCV-#6, the vaccinee showed protection. Neutralization of HCV-#6 with the antiserum from this protected vaccinee was achieved by inoculation of this mixture into another chimpanzee. These results suggest that vaccination with a peptide-vaccine of homologous HVR1 is effective in the chimpanzee.

Characteristic loss of heterozygosity in chromosome 3P and low frequency of replication errors in sporadic renal cell carcinoma.

PURPOSE: A high frequency of genetic loss at 4 loci on chromosome 3p has been shown in human sporadic renal cell carcinomas (RCCs), but the relative contribution of each locus is not well known, and the involvement of DNA replication errors (RERs) in carcinogenesis of RCCs remains unclear. We report the simultaneous comparison of genetic loss at the 4 chromosome 3p loci and RERs in sporadic RCCs. MATERIALS AND METHODS: DNA was extracted from 33 Japanese sporadic RCC samples, and examined for loss of heterozygosity (LOH) and RERs by amplification of 14 microsatellite regions. LOH of the von Hippel Lindau (VHL) gene was analyzed by a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. The target sequences of RER, transforming growth factor beta type II receptor (TGFbetaRII) and Bcl-2-associated X protein (BAX) genes were amplified and analyzed for mutations by sequencing. RESULTS: LOH of the VHL gene was observed in 53.3% of RCCs, a higher frequency than that of the 4 regions 3p12-p13 (18.8%), 3p14.2 (17.4%), 3p21 (21.2%) and 3p25-p26 except for VHL (31.3%). There were no RERs in 14 microsatellite regions, including the mononucleotide (A)10 repeats of the TGFbetaRII gene and (G)8 repeats of the BAX gene. CONCLUSION: Japanese sporadic RCCs were characterized by predominant loss of VHL gene and low contribution of the other 3 candidate RCC tumor suppressor genes. RERs, mostly caused by a defect of DNA mismatch repair, might only rarely be involved in the carcinogenesis of sporadic RCCs.

Multiple sequence-reactive antibodies induced by a single peptide immunization with hypervariable region 1 of hepatitis C virus.

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is known to contain neutralizing epitopes. We previously found that murine antibodies against HVR1-#6 captured a different isolate, HCV-#7, and cross-reacted with the HVR1 peptide of HCV-#7. We investigated the inducibility and generality of cross-reaction of animal anti-HVR1 antibody responses in this study. Anti-HVR1-#7 antibodies, which were induced in mice and a chimpanzee by immunization, were found to be cross-reactive to HVR1-#6 peptide. Antibody responses against HVR1-#6-1 and HVR1-#7 peptides were detected in 11/165 (6.7%) and 26/165 (15.8%) HCV-infected individuals, respectively. Nine HVR1 sequences from six individuals, who were strongly positive for anti-HVR1-#7 antibodies, were only 50-64.5% identical to that of HVR1-#7. All nine of these HVR1 peptides were reactive to sera from the six patients and/or to antisera against HVR1-#6 and HVR1-#7 produced in mice and chimpanzees. Cross-inhibition tests of chimpanzee antisera indicated that a given species of anti-HVR1 antibodies was reactive to multiple HVR1 sequences. Fine epitope mapping of polyclonal and monoclonal anti-HVR1 antibodies showed that conserved subregions in HVR1 sequences determined the observed immunological cross-reactivity. Our data demonstrate that cross-reacting anti-HVR1 antibodies are inducible by a single peptide immunization.

Two types of sporadic multiple colorectal cancers with and without HNPCC-like genetic instability.

BACKGROUND/AIMS: Sporadic multiple colorectal cancers (MCCs) potentially have similar genetic alterations to hereditary nonpolyposis colorectal cancers (HNPCCs), but genetically unstable MCCs other than HNPCCs are not well characterized. We report the frequency of HNPCC-like sporadic multiple colon cancers and their characterization as for HNPCC-related gene mutations. METHODOLOGY: Microsatellite instability (MSI) at 12 microsatellite loci was examined by a polymerase chain reaction in 19 cases each of MCC and single colorectal cancer (SCC). The target sequences of MSI, transforming growth factor beta type II receptor (TGF beta RII) gene, and the HNPCC genes, hMSH2 and hMLH1, were amplified and analyzed for mutations by sequencing. RESULTS: In 5 of 19 cases with MCC, MSI was observed at more than 4 microsatellite loci, and the other cases including all SCCs showed no MSI or MSI(+) at a single locus. In 4 of the 5 severe MSI(+) cases, a 10-bp adenine tract at codons 125-128 of the TGF beta RII gene was mutated. In terms of the hMSH2 and hMLH1 genes, only silent mutations and non-critical amino acid substitutions were found. CONCLUSIONS: We found severe MSI in 26% of sporadic multiple colorectal cancers. Mutations of the TGF beta RII gene are closely associated with severe MSI(+) MCCs as observed in HNPCC, suggesting that these MCCs develop by the similar carcinogenic process to HNPCC.

Murine antibodies against E2 and hypervariable region 1 cross-reactively capture hepatitis C virus.

The absence of readily available animal and cell culture models for hepatitis C virus (HCV) replication has bottlenecked research on protective immunity to HCV infection. Antibodies reactive with HCV virions in vitro are assumed to be candidates for neutralizing or inhibitory antibodies against HCV. To find potentially neutralizing or inhibitory antibody candidates, anti-C, anti-E1, anti-E2, and anti-HVR1 antisera acquired from mice immunized with corresponding recombinant proteins or synthetic peptides were used to capture HCV viral particles in vitro based on antibody-virus interaction assays. Both anti-E2 and anti-HVR1 antibodies effectively captured HCV in vitro. Furthermore, it was found that anti-E2 and anti-HVR1 antibodies could immunoprecipitate an isolate of HCV unrelated to the original antigenic HCV isolate. ELISA confirmed that anti-HVR1 antibodies cross-reactively bind to these unrelated HVR1 peptides. These findings suggest that anti-E2 and anti-HVR1 antibodies induced in mice have the ability to bind with HCV particles in an isolate cross-reactive manner and highlight the possible application of combining several sequences of HVR1 to generate broadly reactive anti-HVR1 antibodies.

Replication error frequencies in primary hepatocellular carcinoma: a comparison of solitary primary versus multiple primary cancers.

AIMS/BACKGROUND: Replication errors (RERs) at microsatellite loci are associated with the development of not only hereditary nonpolyposis colon cancer but also sporadic cancers. To examine the association between RERs and human hepatocarcinogenesis, we looked for microsatellite instability in solitary and multiple primary hepatocellular carcinomas (HCCs). METHODS: DNAs were extracted from 34 solitary primary HCCs and 14 HCCs with multiple primary cancers. Twelve microsatellite alleles were amplified by PCR from the DNAs, and RERs were assessed by their mobility shift. RESULTS: RERs were found in only two cases (6%) of solitary HCC and four cases (29%) of HCC with multiple primary cancers. Two of the four HCCs with multiple primary cancers showed RERs at two and three microsatellite loci, respectively. Of 12 microsatellite loci examined, TP53 and D16S476 sensitively detected RERs in HCCs with multiple primary cancers, at a frequency of 23% and 33%, respectively. CONCLUSIONS: RERs are rarely associated with carcinogenesis in human primary HCC, and RER-related genetic alterations may be associated with a part of HCC with multiple primary cancers. If future studies confirm this association, then the two probes TP53 and D16S476 may be useful for the prediction of development of multiple primary cancers with HCC.

Genetic and serological evidence for multiple instances of unrecognized transmission of hepatitis C virus in hemodialysis units.

We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.

Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese.

The nitric oxide synthase (NOS) gene is thought to be associated with essential hypertension (EH), because NO is implicated in endothelium-mediated vasodilation. We investigated the possible association between the alleles of simple tandem repeat DNA polymorphism of the endothelial constitutive NOS (cNOS) gene and EH in Japanese subjects. In all, 100 patients with EH and 123 subjects with normal blood pressure were studied. Polymerase chain reaction was used to amplify the CA repeat site in the endothelial cNOS gene and alleles based on the CA repeat number were determined. The allele frequencies in the hypertensive group and normotensive group were then compared. Twenty-three alleles were identified in this study of Japanese subjects. The overall distributions of allele frequencies in the two groups were not significantly different. However, comparing the allele frequencies in the EH group without left ventricular hypertrophy (LVH) and the normotensive group, the overall distributions were significantly different (p = 0.019). The 33-repeat allele was found more frequently in the EH group without LVH than in the normotensive group (p = 0.000047, Odds ratio = 3.71). In conclusion, the 33-repeat allele of the endothelial cNOS gene is associated with EH without LVH, and may be a genetic marker of EH in Japanese subjects.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti-HCV assays, we studied 59 anti-HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti-HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C-terminal truncated soluble E2 (sE2) protein (a.a. 390-683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti-HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti-HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti-HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region.

Paternity testing by the detection of D1S80 VNTR using fluorescence image analyzer (Dualcolour system).

An improved method for DNA polymorphism typing of D1S80 VNTR locus and its application to paternity testing are described. For accurate estimation of the length of polymorphic DNA fragments, the size marker was labeled with fluorescence different from that of PCR primers, and co-electrophoresed as an internal standard. The dualcolour system of fluorescence image analyzer was used to detect the fragments and determine their size. This internal marker method could successfully overcome the problems of band pattern distortion and tailing, besides it allows easy and accurate interpretation of the DNA types. Our results indicate that the internal marker method is much more accurate than the method of using size marker in gel, even with the presence of distortion or tailing of the band patterns. Family studies applying this method showed complete agreement between the observed and predicted types.