Am JHum Genet 31:601-619, 1979

Genetic Linkage between Hereditary Hemochromatosis and HLA K. KRAVITZ,1 M. SKOLNICK,' C. CANNINGS,1'4 D. CARMELLI,' B. BATY,1 B. AMOS,2 A. JOHNSON,2 N. MENDELL,2 C. EDWARDS,3 AND G. CARTWRIGHT3

SUMMARY A large Mormon pedigree of a proband with hemochromatosis was studied, using transferrin saturation as the quantitative phenotypic trait. The analysis indicated that the inheritance of hemochromatosis was recessive, with partial expression in some heterozygotes. The lod score of 6.88 (0 = .0) was strongly indicative of linkage between the hemochromatosis locus and the human major histocompatibility (HLA) loci.

INTRODUCTION

The mode of inheritance of hemochromatosis has generated considerable dispute. Different genetic transmissions have been advocated: dominant [1-8], recessive [9, 10], intermediate (i.e., recessive with partial expression in heterozygotes) [11- 14], polygenic [15], and multifactorial [15, 16]. The suggestion has also been made that hemochromatosis may result from any one of a group of diseases, each with a different mode of inheritance [17]. Although most studies favor the dominant hypothesis, many have drawn conclusions from small families and have failed to do any rigorous model fitting. Furthermore, many who have concluded dominant inheritance have based their conclusions on the segregation of minor abnormalities in pedigrees. An equally plausible explanation of Received December 21, 1978; revised May 7, 1979. This research was supported by grants AM-20630, CA-16573, GM-10367, GM-07464, RR-05428, and FR-00064 from the National Institutes of Health, and a grant to Dr. Edwards from the Harry E. Carleson Foundation. 1 Department of Medical Biophysics and Computing, University of Utah Medical Center, Salt Lake City, UT 84132. 2 Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710. 3Department of Internal Medicine, University of Utah Medical Center. Department of Probability and Statistics, University of Sheffield, S3 7RH, United Kingdom. 1979 © by the American Society of Human Genetics. 0002-9297/79/3105-0013$01.93

601

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KRAVITZ ET AL.

these data is an intermediate form of inheritance [11, 12] in which the homozygotes exhibit clinically manifest disease, while heterozygotes exhibit only minor iron abnormalities. Data from the two largest studies [10, 13], 96 and 106 families, respectively, support an intermediate rather than a dominant mode of inheritance. The discovery of an association of specific histocompatibility (HLA) antigens with hereditary hemochromatosis [ 18] offers promise to resolving the mode of inheritance of this disease. The original observation by Simon et al. [18] has been confirmed in France [15], Ireland [19], Scotland [20], England [21], and Germany [22]. One study [15] demonstrated that the frequency of HLA-A3 (78.4% vs. 27.0%) and HLA-B14 (25.5% vs. 3.4%) was significantly higher in 51 affected individuals than in a control group. McDevitt & Bodmer [23, 24] suggest that the associations between HLA and certain diseases are most likely due to tight linkage between the HLA loci and the loci which control susceptibility to those diseases. The observation of these associations in a number of different populations makes it unlikely that the associations were caused by effects of population structures. Furthermore, the fact that the associations were found between hemochromatosis and a number of distinct HLA antigens at both the HLA-A and -B loci decreases the likelihood that a specific antigen directly promotes the development of disease. Simon et al. [25] reported linkage between HLA and hemochromatosis which is compatible with a recessive form of inheritance. On the other hand, Muir et al. [8] reported linkage and concluded a dominant mode of inheritance. As was the case in many studies which concluded dominant inheritance, the authors assumed that minor iron abnormalities represented latent disease. Thus, their data may also have been compatible with an intermediate form of inheritance, assuming that the individuals with minor abnormalities were simply heterozygotes who will not develop overt disease. This paper describes the study of a large Utah Mormon pedigree of a proband with hemochromatosis. We investigated the mode of inheritance, compared possible major gene models, and evaluated these models using recently developed methodologies [26, 27]. We demonstrated linkage between HLA and hemochromatosis, assigned probabilities of genotypes to pedigree members, and described the initial estimates of iron profiles of each genotype. A preliminary discussion of our results has been presented elsewhere [14, 28]. MATERIALS AND METHODS

Clinical Material All individuals and patients studied were members of a large Mormon pedigree (fig. 1) previously designated Pedigree A [29]. The basic pedigree included descendants of the first generation couple, their spouses, and three sets of spouses' parents. This basic pedigree structure was used for all model-fitting procedures. A supplement including the siblings of two of the three aforementioned spouses was utilized to clarify risks. There was no known consanguinity. None of the individuals was an alcoholic, and most consumed no alcohol because of religious convictions. None had received oral or parenteral iron or blood transfusions. Several had donated blood at sporadic intervals in the past, but none was a professional blood donor. Serum iron and transferrin saturation were measured on 181 individuals. Typing of the HLA-A and -B loci was performed on 118 individuals by a standard two-stage microlymphotoxicity dye

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exclusion technique [30]. Typing of the C and D loci was performed on a few selected individuals as was typing of B-cell lymphocytes to determine if haplotypes identical in the HLA-A and -B loci were in fact identical after more complete typing [31]. Urinary iron excretion after deferoxamine was measured on 54 individuals, serum ferritin concentration on 156, and liver biopsies were performed on 27. Classification of Iron Status The iron status of individuals was evaluated by measuring the following: serum iron concentration, transferrin saturation, urinary iron excretion after administration of deferoxamine, serum ferritin concentration, hepatic iron concentration, and grade of stainable iron in hepatic parenchymal cells. Methods used for these measurements and the values obtained in a normal population have been reported previously [29]. The following definitions of major and minor iron load were used as a description of iron profile for the pedigree drawing (fig. 1). The definition of major iron load was also used to define the set of affected individuals for estimating parameters (Z, W) of the disease threshold (see Models section). A major iron load was defined as being present in those individuals with a serum iron greater than 170 ,ug/100 ml, transferrin saturation greater than 70%, urinary iron excretion after deferoxamine greater than 3.0 mg/24 hr, serum ferritin greater than 1000 ng/ml, hepatic iron concentration greater than 300 ,ug/100 mg wet liver, and a hepatic parenchymal cell stainable iron of grade 4. Individuals with a major iron load required more than 40 phlebotomies (500 ml each) before depletion of the excess iron. Seven patients in this pedigree met all of these criteria. A minor iron load was defined as a transferrin saturation greater than 50% and/or a hepatic iron concentration greater than 25 ,g/100 mg wet liver, but less than 300 ,ug/100 mg (providing the iron was deposited in hepatic parenchymal cells and not in Kupffer cells). Twenty-nine individuals were identified as having a minor iron load. Five individuals with a minor iron load required less than 16 phlebotomies (500 ml each) before depletion of the iron excess. HLA Typing Initial characterization was made using a panel of 123 sera to test for the HLA-A and -B specificities defined by the 1975 Workshop, except for Aw36 but including newer specificities of the 1977 Workshop: Bw44, Bw45, Bw5l, Bw52, and Bw53 [32, 33]. The procedure used was the NIH method as modified by the addition of a wash step [34]. In addition, 40 selected members of the pedigree were retyped using the panel of sera from the 1977 International Workshop to characterize HLA-A, -B, and -C specificities. The procedure for HLA-A, -B, and -C was the standard NIH technique [30]. In addition, 38 members were characterized for B-cell antigens using the F (abl)2 separation method for the one-hour-plus-one-hour-incubation procedure of the Duke-Durham VA laboratory [35]. This procedure permits the identification of all presently recognized DRw specificities and those local specificities designated DuB groups. HLA Haplotyping The HLA haplotypes of the proband were A3-B7 and A29-B12. The 29-12 haplotype, designated X in figure 1, was introduced into the pedigree by II- 13. The father and uncle of the proband carried only HLA antigens A3 and B7, leading to the supposition that they were A3, B7 homozygotes. This was confirmed when the B-cell (HLA-DR) antigens allowed the identification of two distinct 3-7 haplotypes, designated A and B (fig. 1). However, B-cell antigens were not identified in all individuals who carried one of these 3-7 haplotypes. Thus, the 3-7 haplotypes which were either A or B are designated H in figure 1. Two additional 3-7 haplotypes were associated with abnormalities in the family. One of these (C) was introduced by III-18 and another (D) by IV-65. These 3-7 haplotypes (A, B, C, or D) in combination or together with the 29-12 haplotype (X) were found in all but one subject with a major iron load. The exception, a spouse (111-40) whose haplotypes were A2-B15 (1) and A26-B27 (J), had a major iron load

n(Z)i

HEMOCHROMATOSIS AND HLA

605

requiring 40 phlebotomies. None of her descendants in generation IV had a major iron load, and none in generation V showed any iron abnormalities. Therefore, her haplotypes did not contribute additional information to the linkage analysis and were not separately considered. Two (or perhaps three) additional 3-7 haplotypes were discovered in the pedigree. One of these (E) was introduced by the same woman (II-13) who contributed the abnormal 29-12 haplotype X. No abnormalities were found in her offspring who carried the E haplotype. The 3-7 haplotype designated F was introduced by 111-46. This individual andthree of her offspring were the only ones in the pedigree to carry this haplotype, and all four werd normal. The final 3-7 haplotype (G) may have been the same haplotype as A or B. This haplotype was found in the offspring of II-16 and II-17 and was associated with some minor abnormalities. Three additional 29-12 haplotypes were present in the pedigree. These were introduced by IV-93, III4-9, and IV- 103 and were designated W. Y. and Z, respectively. Models Transferrin saturation was chosen as the quantitative trait used in the analysis. The following considerations dictated that choice. Urinary iron excretion and liver biopsy data were not chosen because they were available only on a small subset of the pedigree (54 and 27 members, respectively). Serum ferritin concentration was undesirable, being markedly influenced by the presence of chronic diseases other than hemochromatosis. Furthermore, serum ferritin levels increase at a later stage of iron loading than do the serum iron and transferrin saturation [5, 36], making it an unreliable indicator in early stages. Finally, serum iron is reported to be less informative in identification of iron load than transferrin saturation [6, 37]. Therefore, transferrin saturation was the logical choice. All blood specimens were drawn at 8 AM in the fasting state, and at least three determinations were made one week or more apart. To reduce the variance of the measure and increase its resolving power, an average of these determinations was used as the estimate of transferrin saturation. The model describing the inheritance of transferrin saturation level is the generalized major locus with two alleles. The phenotypic expression of each genotype is assumed to have a normal distribution, with mean and variance (,ui, ai2), for genotype i. The three genotypes, determined by a one-locus two-allele model, are: (1) abnormal homozygote, (2) heterozygote, and (3) normal homozygote. The validity of the normality assumption is tested later. Transferrin saturation is assumed to relate to hemochromatosis through a liability threshold model [38-40]. Using Morton's notation [38], the liability is w = x + k, where x is the transferrin saturation, and k is a normally distributed environmental effect with mean zero and variance W. The threshold, Z, is defined such that an individual is affected if w > Z. Z and W were estimated, using the definition of a major iron load give above as the disease classification. The likelihood function for estimating Z and W is

where

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D

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Z-x (DVSJ

KRAVITZ ET AL.

606

where Z and W are the maximum likelihood estimates of Z and W, obtained from the likelihood function given above. The proportion of individuals in the population within each genotypic class is assumed to be in Hardy-Weinberg equilibrium for both the marker and the disease locus. In the linkage analysis, we did not attempt to estimate the magnitude of the postulated disequilibrium between the disease locus and HLA haplotypes in this population. Thus the haplotype frequencies used were calculated by taking the product of the two "allele" frequencies. The HLA-A and -B loci were considered as a single locus, as they are tightly linked and segregate together in this pedigree. Three HLA haplotypes were defined: A3-B7, A29-B12, and "all others pooled." The frequencies of the 3-7 and 29-12 haplotypes used were .055 and .0065 respectively [41]. The frequency of the hemochromatosis gene was held constant at .01 during the maximization procedure, because single pedigrees do not provide reliable estimates of gene frequency. It was calculated by taking the square root of the estimated disease frequency, .0001 [42]. Lod score calculations were made across a range of gene frequencies (.01 . q s .09) to test the sensitivity of the result. Ascertainment The pedigree was originally ascertained through a single individual with hemochromatosis. The subsequent collection of the pedigree proceeded sequentially, with interesting sections being further investigated. This sequential sampling of a pedigree under single ascertainment does not require any modification of the likelihood [43]. However, a correction is needed because the proband was determined by disease status, which is correlated to the character (transferrin saturation) under investigation. The unequal probabilities of sampling the various possible pedigrees are due to the unequal numbers of affected individuals in the various pedigrees within the population [44, 45], and need correction. However, in this case, the correction can be made in a simple manner. Our refinement is to compute the likelihood, L, of the model where L = P(Observations Model n Proband diseased), and the observations consist of the pedigree structure, the individuals' phenotypes, and the proband's identity. It can then be proved [46] that L

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L

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P(PhenotypesiPedigree n Model) Disease frequency

The disease frequency is calculated, using the estimates 1, W, and the (,mj, 3rj)'s, as

P(Proband diseased) = ± p ¢(

\

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where pi is the Hardy-Weinberg frequency of genotype i. Age and Sex Effects The clinical expression of hemochromatosis is affected by both age and sex of the individual, so both age- and sex-effects in the data must be considered. Figure 2 shows transferrin saturation levels plotted against age for each sex. There is clearly no significant correlation between transferrin saturation level and age for females (r = -.02). In males, there is a slight positive trend (r = .24) which accounts for about 6% of the variance and appears to be largely due to a

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Pedigree Analysis Our basic objectives were to fit a genetic model for the transmission of transferrin saturation level, to estimate the disease penetrance, and to investigate possible linkage with the HLA loci. We did this by comparing likelihoods for various models. The likelihoods were calculated using the transferrin saturation values as the phenotype. We utilized a maximum likelihood procedure to obtain estimates of the mean and variance of the transferrin saturation distribution within each genotype.

A comparison was first made between a dominant, a recessive, and an intermediate model. In the intermediate model, the mean of the distribution of transferrin saturation in the heterozygotes was between that of the two homozygotes. To compare the three models, the maximum likelihood procedure was utilized three times, each with a different set of initial parameters. The HLA data were excluded for these calculations. The initial parameters represent the dominant, recessive, and intermediate models (table 1). A sample of normal individuals studied in Utah showed normal values of transferrin saturation closely centered about a mean of 35%, with a standard deviation of 7.5% [47]. These values were used as initial estimates of the mean and

KRAVITZ ET AL.

608

TABLE 1 INITIAL CONDITIONS: MEANS AND STANDARD DEVIATIONS OF PHENOTYPIC DISTRIBUTIONS WITHIN EACH GENOTYPE ABNORMAL HOMOZYGOTE INITIAL CONDITIONS

Dominant ................... Recessive ................... Intermediate .................

85.0 85.0 85.0

HETEROZYGOTE

NORMAL HOMOZYGOTE

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M2

M3

03

15.0 15.0 7.5

85.0 35.0 45.0

15.0 7.5 10.0

35.0 35.0 35.0

7.5 7.5 7.5

standard deviation of the distribution for the normal homozygote genotype. Initial values for the other two genotypic distributions were estimated from the data (fig. 3). Calculation of the likelihoods is achieved using the methodology of Cannings et al. [26, 27], in which an algorithm for the successive application of Bayes's theorem to subsections of the pedigree is developed. Similar approaches in human genetics are credited to Morton [44, 45] for nuclear families: Heuch & Li [48], Elston & Stewart [49], and Ott [50] for pedigrees of a restricted structure; and Lange & Elston [51] for general pedigrees. Here we describe the aspects of the algorithm relevant to the present study, including specific details of the analysis of the linkage between a qualitative marker and a quantitative trait. We calculate [52, 53] the likelihood, L, of a model, given the observations, where L = P(Observations Model n Parameter values n Gene frequencies). The specification of the model requires a penetrance function and a transmission model. The penetrance function, Pen(xli), is defined by Pen(xli) = P(Phenotype is xI Genotype is i). In this context, x is the transferrin saturation level. The three genotypes are abnormal homozygote, heterozygote, and normal homozygote, as determined by a one-locus two-allele model. Thus, under the assumption of a normally distributed phenotypic expression of the genotype, Pen(xli) = O (x), where

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exp

-

( cji2/2

and ,ui and o-i are the mean and standard deviation of the phenotypic expression of genotype i. The transmission model, in this case simple Mendelian inheritance, specifies the probability that parents with a specific genotype will have an offspring of a particular genotype. Thus Trans(ilj,k) = P{Offspring has genotype ilParents have genotypes j and k). The specific values are for a single, diallelic locus under Mendelian segregation.

Risk Analysis Risks (i.e., probabilities of genotypes) were calculated for individuals in the pedigree, incorporating information in three different combinations. The three calculations use the maximum likelihood estimates of the parameters (Aui, on); the transferrin saturation levels of the pedigree members; and (i) no additional information, (ii) pedigree without HLA, or (iii) pedigree with HLA. The probabilities for (i) are computed for each individual as gj= Pi i (x) i

3

E Pj 4kj (x)

where gi is the probability of being genotype i for the individual; pi is the frequency of genotype i, assuming Hardy-Weinberg equilibrium; and x is the individual's transferrin saturation level. The probabilities for (ii) and (iii) are computed, using the likelihood algorithm described above as

HEMOCHROMATOSIS AND HLA

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= P{Observations n Individual is genotype i IModel}

P{ObservationslModel} where the phenotypic observations include the information specified above. RESULTS

Fitting the Model The maximum likelihood estimates of the threshold, Z, and the environmental liability variance, W, were Z = 71, and W = 13 (individuals V-60 and V-82 were too young to express the disease and were excluded from this procedure). Table 2 summarizes the results of the maximization procedures for the three initial parameter sets without the HLA data. Also shown are the disease incidences within each genotype, which were calculated using the maximum likelihood estimates of ui, oi, Z, and W. Two distinct local maxima were observed on the likelihood surface, with similar likelihood values. These corresponded to intermediate models which were, in one case, close to a recessive model (d = .17), and in the other, to an additive model (d = .61), where d is the degree of dominance [38], defined as (J.2 - /3)/(l1 -3)We have not assumed equal variances, and thus use the degree of dominance concept only to describe the two models. Both models indicate a gene dose effect. In one case (d = .61), the mean of the heterozygote distribution is nearly midway between the means of the two homozygote distributions. However, the variance of that heterozygote distribution is much larger than the other two. This gives heterozygotes a high probability of expressing the disease, and tends to make the model approach a dominant model with partial penetrance in the heterozygote. In contrast, the other local maximum (d = .17), represents a model in which few heterozygotes express the disease. Linkage Analysis Table 3 gives the log likelihoods and lods for various values of 6, the recombination fraction for linkage of the major locus with the HLA loci. These were calculated holding the /, &)'s constant at the two sets of values in table 2. At one local

KRAVITZ ET AL.

610

TABLE 2 LOCAL MAXIMUM LIKELIHOOD ESTIMATES FOR THREE INITIAL CONDITIONS WITHOUT HLA DATA ESTIMATED PARAMETER INITIAL CONDITIONS

Dominant U .................... a ................... Incidence .............. Recessive and intermediate , ................... ..................

Incidence ............

IN

TABLE 1,

VALUES

Abnormal homozygote

Heterozygote

Normal homozygote

85.0 15.0 .816

65.3 22.0 .397

34.8 11.0 .001

87.2 10.2 .932

41.8 12.8 .014

32.8 10.2 .000

LIKELIHOOD

DEGREE OF DOMINANCE

-292.9

.61

-292.7

.17

LOG

)

maximum (d = .17), the highest likelihood was obtained for 6 = 0, indicating close linkage. Conversely, at the other local maximum (d = .61), there was no evidence for linkage. Accordingly, a new set of maximum likelihood estimates were obtained for the (Ii, o-j)'s, including the HLA data with 6 fixed at .0. The initial values used for the (pi, &i)'s were those of both local maxima in table 2. Table 4 gives the new maximum likelihood estimates, which correspond closely to the d = .17 case. Another lod score was calculated by using the new maximum likelihood estimates of the (pi, ui)'s shown in table 4. This lod score was equal to the log of the ratio of the following two likelihoods: (1) the maximum likelihood with respect to all parameters, including 6, and (2) the maximum likelihood with respect to all parameters except 6, which was held constant at .5. This lod score was calculated to be 5.01 (6 = .0) and was similar to that shown in table 3 (d = .17). A recalculation of the lod, using the supplemented pedigree, yielded a lod score of 6.88. To see how the lod score was affected by gene frequency, lods were calculated for gene frequencies ranging from .01 to .09. The maximum variation in the lod across this range was 3%. The lod score was thus insensitive to gene frequency. Risk Analysis Figure 4 shows the number of people in the pedigree who fall into each risk category for each of the three methods of computing risks. The two sets of risks calculated using the pedigree data show more certainty (i.e., fewer persons with risks in the .3 - .7 range) in defining risks than do those calculated using transferrin saturation alone. Furthermore, the risks calculated using HLA data were more clearly defined. Risks were also used to calculate the means and variances, by genotype and sex, of all six measures of iron status (table 5). In this case, we used weighted means, the weights being the genotypic risks calculated using the HLA data (iii). As a partial check on the validity of the assumption of normality, the risks calculated for the pedigree members by method (iii) were utilized in the following manner. Frequency tabulations for transferrin saturation level were made for each genotype, each individual contributing to each tabulation with a weight equal to the appropriate

HEMOCHROMATOSIS AND HLA

611

TABLE 3 LODS AND LOG LIKELIHOODS FOR THE Two LOCAL MAXIMA IN TABLE 2 RECOMBINATION FRACTION 0 DEGREE OF DOMINANCE

LOG LIKELIHOOD

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.0

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-

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(0 = .5)

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- 0.56 2.98

- 0.24 1.99

-0.06 0.94

- 331.5 - 331.3

risk. Probit plots are shown in figure 5. The fit appears satisfactory. No formal goodness of fit test was used, because the dependence between the individuals with respect to the probabilities of each genotype would invalidate any known procedure. DISCUSSION

The mode of inheritance of hemochromatosis appears to be recessive, with heterozygotes showing a partial biochemical expression. The analysis indicates that two copies of chromosome 6, each carrying an abnormal gene, are highly penetrant for the full expression of the disease. Vertical transmission of abnormalities appeared across four generations in the pedigree (from II-13 to V-82) with one generation skipped (111-27). The iron status of the individual skipped, a 61-year-old female, could not be adequately evaluated because of her severe rheumatoid arthritis, which had been treated for many years with aspirin and prednisone. A liver biopsy could not be performed, and the rheumatoid arthritis may have influenced her transferrin saturation value. Without the HLA data and the extended pedigree, this section could have- been used as a demonstration of a dominant mode of inheritance. Those who have concluded that transmission is dominant have interpreted the vertical transmission of minor iron abnormalities as latent disease. In fact, many of these studies have failed to find clinically manifest disease in more than one generation [16] and have based their conclusion of dominant inheritance solely upon this assumption. Evidence from this study refutes this assumption. There are many individuals in generations II, III, and IV of this pedigree (fig. 1) who are old enough to express the disease, yet they show only minor iron abnormalities. Clearly, a dominant model does not fit this pedigree data. Other studies have provided further evidence to refute the assumption that minor TABLE 4 REMAXIMIZED ESTIMATES, USING HLA DATA WITH 6 FIXED AT 0.0 Abnormal homozygote ......................

C.

Incidence

87.5 10.3

.0.933

Heterozygote

Normal homozygote

41.0 11.9 0.008

32.0 9.8 0.000

Log likelihood

X

326.7

Degree of dominance 0.16

KRAVITZ ET AL.

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FIG. 4.-Histogram of genotypic risks for individuals whose HLA types were known (black), and unknown (white). Three sets of risks were calculated, using transferrin saturation data, (J2,, &i)'s, and: (i) no additional data, (ii) pedigree structure, or (iii) both pedigree structure and HLA data.

abnormalities represent early stages of disease. One study [54] directly tested this assumption by following the progress of three asymptomatic pedigree members who displayed minor iron abnormalities. All three individuals, two daughters and a granddaughter of a hemochromatosis proband, failed to show evidence of progression of the disease. In a study of 96 families, Saddi & Feingold [10] found no significant difference in unsaturated iron binding capacity (UIBC) between the parents and offspring of hemochromatosis probands. Likewise, Simon et al. [ 13] found no significant difference in UIBC or serum iron between the parents and offspring of 106 hemochromatosis probands. Although no consanguinity was discovered in this pedigree, the consanguinity reported in previous studies [1, 10, 13, 55-58] supports a recessive hypothesis. One family study [58] reported that a 55-year-old female proband and her affected brother were offspring of an uncle-niece mating. Debre et al. [1] found one first-cousin marriage out of 36 families of hemochromatosis probands, corresponding to a coefficient of consanguinity [59] of 174 x 1 0-5. Simon et al. [ 13] reported a coefficient of consanguinity of 271 x 10-5, corresponding to three first-cousin marriages and one

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