Abstract

We recently described an untranslated CTG expansion that causes a previously undescribed form of spinocerebellar ataxia (SCA8). The SCA8 CTG repeat is preceded by a polymorphic but stable CTA tract, with the configuration (CTA)1–21(CTG)n. The CTG portion of the repeat is elongated on pathogenic alleles, which nearly always change in size when transmitted from generation to generation. To better understand the reduced penetrance and maternal penetrance bias associated with SCA8 we analyzed the sequence configurations and instability patterns of the CTG repeat in affected and unaffected family members. In contrast to other triplet repeat diseases, expanded alleles found in affected SCA8 individuals can have either a pure uninterrupted CTG repeat tract or an allele with one or more CCG, CTA, CTC, CCA or CTT interruptions. Surprisingly, we found six different sequence configurations of the CTG repeat on expanded alleles in a seven generation family. In two instances duplication of CCG interruptions occurred over a single generation and in other instances duplications that had occurred in different branches of the family could be inferred. We also evaluated SCA8 instability in sperm samples from individuals with expansions ranging in size from 80 to 800 repeats in blood. Surprisingly the SCA8 repeat tract in sperm underwent contractions, with nearly all of the resulting expanded alleles having repeat lengths of <100 CTGs, a size that is not often associated with disease. These en masse repeat contractions in sperm likely underlie the reduced penetrance associated with paternal transmission.

Received 12 April 2000; Revised and Accepted 28 June 2000.

INTRODUCTION

Trinucleotide repeat expansions are the underlying genetic mechanism responsible for a number of inherited neurological diseases, including fragile X mental retardation (FMR), myotonic dystrophy type 1 (DM1), Huntington’s disease and seven forms of dominantly inherited spinocerebellar ataxia (SCA) (13). In contrast to the CAG expansions encoding glutamine tracts that are responsible for SCA1, -2, -3, -6 and -7, we have recently reported that an untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8) (2). SCA8 and DM1 are the only identified diseases caused by pathogenic CTG repeat expansions that are transcribed but not translated into proteins (1,2).

Although the SCA8 CTG expansion is similar to that for DM1, the genetic instability and reduced disease penetrance of SCA8 differ distinctly from DM1 and other trinucleotide repeat disorders (1,2,4). In a large SCA8 family (MN-A) that we have characterized (Z = 6.8, θ = 0.0) affected individuals have longer CTG repeats (107–127, mean 117) than 21 unaffected expansion carriers (mean 90) (P = 1 × 10–8) (4). The reduced penetrance of the disease can sufficiently obscure the pattern of inheritance so that affected individuals can appear to have sporadic or recessively inherited ataxia even though the SCA8 expansion on chromosome 13q21 is dominantly inherited.

Although detailed clinical evaluations of the large MN-A family have allowed us to establish a pathogenic size threshold for the MN-A family (4), the repeat size range found in affected members of 11 different ataxia families that we have characterized is much broader, ranging from 71 to 800 CTG repeats. Usually, however, alleles with <100 CTG repeats and >250 CTG repeats are non-penetrant. Although the SCA8 expansion is transmitted from both males and females, among the 11 different ataxia families 26 of 30 affected individuals inherited maternally transmitted expansions. The maternal penetrance bias is consistent with a preponderance of repeat expansions during maternal transmission that result in alleles in a size range (100–250 CTGs) associated with higher disease penetrance, whereas paternal contractions usually result in shorter, less pathogenic repeats (<100 CTGs).

The reduced penetrance of SCA8 as well as the adjacent highly polymorphic (CTA)1–21 repeat tract have made it difficult to establish a pathogenic size threshold that can be applied to all families (2,5). In addition, non-penetrant expansions found both within ataxia families and in rare instances in the general population have caused others to suggest the possibility that the repeat expansion may be a non-pathogenic polymorphism that is tightly linked to another ataxia mutation (2,57). Although the frequency of expansions of >91 combined CTA/CTG repeats among our apparently unrelated ataxia probands (8/102) (2) is significantly higher than in the general population (∼1 in 500 to 1 in 1000) (2,5,6), rare large alleles (600–800 repeats) in the general population appear to occur at higher frequency (∼1 in 1200) than does ataxia (∼1 in 10 000). These data suggest that either: (i) the SCA8 expansion is a non-pathogenic polymorphism in linkage disequilibrium with an ataxia locus; or (ii) the CTG repeat can cause ataxia, but other modifiers, such as repeat length or sequence configuration, affect disease penetrance.

We believe that the following lines of evidence strongly support the conclusion that the SCA8 CTG expansion causes ataxia: (i) the linkage data in the MN-A family (LOD 6.8 θ = 0.00); (ii) the biological relationship between repeat length and disease in the MN-A family with affected family members having longer CTG repeat tracts (mean 116) than 21 asymptomatic expansion carriers (mean 90); (iii) the high frequency of expansions among apparently unrelated ataxia patients (8 of 102); and (iv) SCA8 transcripts are primarily expressed in central nervous system tissue, including cerebellum (2,4).

To better understand the reduced penetrance of SCA8 and the maternal penetrance bias of the disease we have further evaluated the CTG repeat and neighboring sequence in affected and unaffected family members and report here that the CTG repeat tract is highly unstable, with sequence interruptions that can change and duplicate during individual transmissions. We have also evaluated the SCA8 expansion in the sperm of expansion carriers and found that the repeat size differed from that found in blood, with significant repeat contractions that are likely to underlie the reduced paternal penetrance. The highly mutable nature of the SCA8 gene has not been observed at other triplet repeat loci and may play a role in the reduced penetrance of SCA8.

RESULTS

Expanded CTG tracts are often interrupted

The SCA8 CTG repeat is preceded by a stable but polymorphic CTA tract with the overall repeat configuration (CTA)1–21(CTG)n (2,5). The CTG portion of the repeat is elongated on pathogenic alleles and nearly always changes size when transmitted from one generation to the next (2). The various SCA8 allele configurations of unexpanded alleles (≤39 CTG repeats), which were determined by sequencing, are summarized in Figure 1A. In contrast to SCA1, SCA2, FMR and FA (814), triplet interruptions were not observed on SCA8 alleles containing ≤39 CTG repeats (Fig. 1A).

The sequence configuration of expanded alleles associated with ataxia in eight families are summarized in Figure 1B. Three of these families have pure CTA and CTG repeat tracts like those found on the unexpanded alleles, four have triplet interruptions within the CTG repeat tract and one has a CCA interruption within the CTA repeat tract. In three families the CTA or CTT interruptions are located near the 5′ end of the CTG portion of the repeat. The fourth family has both a CTC interruption between the CTA and CTG tracts and four additional CCG interruptions distributed within the first 38 triplets of the CTG tract. The expanded allele from this individual and his son had the same configuration. One of the families with an uninterrupted CTG repeat has a CCA interruption within the CTA repeat tract.

Of 1200 control chromosomes, four individuals with repeat tracts containing ≥45–78 CTGs were found by PCR. Subsequently one very large expansion (∼800 CTGs) was detected by Southern analysis in a CEPH grandparent. Figure 1C summarizes the configurations of three alleles found in the general population with sizes below the range typically associated with ataxia. One of these alleles has two CAG interruptions that are fairly evenly distributed within the CTG repeat, another has two CTA interruptions at the 5′ end of the CTG repeat and the third has two CGG triplets between the CTA and CTG repeat tracts.

Because only four families with CTG tracts >45 repeats had pure repeat tracts like those found on unexpanded alleles (<45 CTG repeats), it appears that long CTG repeat tracts are predisposed to point mutations. All six possible changes from CTG at the second or third positions have been observed on alleles with >45 CTG repeats. Thus far, sequence changes have not been found in the cytosine at the first position of either the CTA or the CTG repeat tract. Most of these interruptions are located near the 5′ end of the CTG tract close to the CTA repeat, suggesting that the juxtaposition of these two repeat sequences may increase the mutability of the repeat tracts. Although the reason that point mutations frequently take place near the border of the CTA repeat is not clear, the length of the CTA repeat does not appear to be related to the occurrence of interruptions.

Duplication of triplet interruptions within the MN-A family

When sequencing expanded SCA8 alleles within the seven generation SCA8 kindred, the MN-A family, we were surprised to find that individuals within the family had different patterns of CCG interruptions within the CTG repeat tract. Haplotype analysis was performed to confirm that the expanded alleles were transmitted from a closely related common ancestor. Although the (CTA)3 repeat tract is stably transmitted, the CCG interruptions within the CTG tract were often duplicated, resulting in six different sequence configurations among the affected and unaffected expansion carriers. Figure 1D summarizes the various allele configurations found and the number of individuals within the family with each repeat configuration (indicated on the right). The segregation of the various alleles within the family is summarized in Figure 2. The presence of uninterrupted CTG expansions in one branch of the family suggests that a least one transmission within the past seven generations resulted in a point mutation changing a CTG triplet to a CCG or in the deletion of a CCG interruption. In two instances duplications of the CCG repeat occurred over a single generation. In case 1 (CTG)5CCG(CTG)101 changed to (CTG)5CCG(CTG)7CCG(CTG)105, whereas in case 2 (CTG)5(CCG)3(CTG)103 became (CTG)5(CCG)4(CTG)87. In other instances repeat duplications that occurred in particular family branches could be inferred. These mutation events altered the CTG repeat configuration at a phenomenally high frequency, with changes occurring at least six times in the 28 transmissions that have taken place in the family since the repeat was transmitted from the common ancestor in generation I. Although all of the affected alleles are interrupted with CCG repeats in the MN-A family, we have found four other ataxia families in which affected individuals have uninterrupted CTG repeat tracts.

En masse contractions of SCA8 repeats in sperm

Southern and PCR analyses were performed on DNA isolated from blood and sperm to further investigate the paternally transmitted contractions in SCA8. Surprisingly, two men with very large SCA8 expansions in blood (500 and 800 repeats) had undergone marked contractions in sperm into narrow size ranges (∼80 and ∼100 CTGs) that less frequently cause ataxia (Fig. 3A). The probe used for the Southern blots did not contain the CTG repeat, therefore, the equal intensities of the bands representing the normal and expanded alleles in the sperm samples indicate that the vast majority of sperm with the expanded allele contain repeats that had contracted to a less penetrant size range (<100 CTGs). Southern analysis of three additional patients with shorter expansions in their blood (80–116) showed a similar contraction trend with a discrete shortening of all or nearly all of the expanded alleles in the sperm DNA (Fig. 3B). Expanded allele sizes found in blood and sperm for the eight men are summarized in Table 1. In all cases the allele sizes in sperm were shorter than in blood and in six of the eight samples all or nearly all of the resulting alleles were in a size range that less often leads to ataxia (below ∼100 CTGs). The en masse contractions in sperm to CTG lengths that are typically less pathogenic are consistent with the reduced penetrance of paternal transmission. The only individuals in which the repeat contractions did not dramatically reduce repeat size in sperm were in a father and son from an unusual family with paternal disease transmission; PCR analysis of sperm samples from these two men demonstrates a discrete shortening of the expanded alleles but to size ranges frequently associated with ataxia (Fig. 3C).

DISCUSSION

Interruptions

During the past decade the characterization of trinucleotide repeat loci has established the strong trend that pathogenic trinucleotide repeat expansions have uninterrupted repeat tracts. The sequence interruptions present on normal SCA1, SCA2, FMR and Friedreich’s ataxia (FA) alleles are thought to stabilize the corresponding repeat tracts and the loss of those interruptions is associated with allele expansion (814). In 1993 Chung et al. (8) reported that 97% of normal SCA1 alleles are interrupted by one, two or three CATs but that expanded alleles have pure, uninterrupted CAG tracts. Similarly, AGG and CAA interruptions are found on unexpanded but not expanded FMR and SCA2 alleles and 5–10 GAGGAA repeats interrupt the normal size GAA tracts in FA (914). The loss of these interruptions enables the resulting pure repeat tracts to form hairpins and other stable secondary structures, which increase the tendency of the overall repeat tract to expand.

In contrast to other triplet repeat diseases in which expanded alleles have fewer or no interruptions, our data suggest that most often normal alleles do not have interruptions and that expanded SCA8 alleles are predisposed to acquiring point mutations. Surprisingly, these interruptions do not confer stability on the trinucleotide repeat but can themselves be duplicated at an extremely high frequency (1 in 5 transmissions in the MN-A family). Both the variety of point mutations observed and the propensity of these interruptions to duplicate result in a diversity of expanded alleles that has not been seen at other trinucleotide repeat loci. Remarkably, six distinct allele configurations are found within the MN-A family alone. Duplicating triplet interruptions observed for SCA8 have not been reported for any of the other trinucleotide expansion loci.

We have found both pure and interrupted SCA8 CTG expansions in ataxia patients. Further experiments are needed to determine what role, if any, the presence of sequence interruptions may play in the reduced penetrance of SCA8 (2,47,15,16).

Duplication models

Experiments performed in yeast (17,18) and Escherichia coli (19) have pointed out the potential for expansions during lagging strand synthesis. These models, in which interruptions are duplicated during DNA replication due to secondary structure, are consistent with the preponderance of duplications that we see; however, other duplication mechanisms, such as the small slipped register model (20), are also possible. Replication-based models for both the CCG duplications as well as changes in 3′ uninterrupted CTG repeat tracts seen in case 1 and case 2 are shown in Figure 4.

We have observed a strong sex bias in SCA8 repeat tract instability, with maternal expansions increasing in size on transmission whereas paternal alleles tend to contract, sometimes dramatically. Because we have observed duplication of interruptions in both a maternal transmission undergoing an expansion and a paternal transmission undergoing a contraction, control of overall repeat length appears to be independent of the duplication of interruptions. In contrast to other triplet repeat diseases, interruptions are frequently introduced and duplicated on expanded SCA8 alleles.

Instability in sperm

Analysis of sperm DNA from men with SCA8 expansions shows a discrete shortening of repeat length in nearly all of the sperm containing the expanded allele. Most often men who are heterozygous for SCA8 expansions in their blood will produce sperm in which ∼50% contain a normal SCA8 allele and 50% have the expanded allele with a CTG repeat in the low penetrance range (<100 CTGs). The equal ratio of mature sperm with normal and expanded alleles seen on the Southern blots suggests that germ cells with smaller expansions are preferentially selected at an early stage of spermatogenesis rather than that mature sperm with larger alleles had died.The dramatic contractions of the SCA8 repeat tract in sperm provide a molecular explanation for the reduced paternal penetrance observed in SCA8. Recently, Silviera et al. (16) suggested that normal SCA8 alleles occasionally expand in sperm. Further experiments will be needed to determine whether the expansions they reported observing by small pool PCR actually lead to an increased frequency of expansions in the general population.

In contrast to SCA8, other dominantly inherited CAG/CTG repeat disorders have a paternal expansion bias with paternal transmission typically generating greater increases in repeat length than maternal transmission (1). Until now the only significant exception to this trend was seen in DM1, in which increases of very large alleles following paternal transmission are less marked than those seen with maternal transmission (2125). The only trinucleotide repeat diseases similar to SCA8 in which a strong bias towards contractions in paternal transmission have been reported are FMR and FA (26,27). In contrast to SCA8, the paternal contraction biases observed in FA and FMR do not have a significant clinical impact on disease penetrance for the following reasons. For FA, although paternal transmission usually results in significant repeat contractions the resulting alleles typically remain within the pathogenic range. For FMR although the repeat contractions observed in sperm of affected males result in alleles below the pathogenic threshold, FMR males are unlikely to have children because of their disease. Although the SCA8 repeat contractions seen in sperm are similar to those observed for the FMR repeat, the dominant inheritance pattern of SCA8 and the relative reproductive fitness of affected males allows for the transmission of a substantial number of SCA8 alleles that are elongated and unstable but within a size range that is not usually associated with ataxia (<100 CTGs). Because the penetrance of SCA8 is affected by the size of the CTG repeat and paternal transmission usually results in shorter alleles that are unlikely to cause ataxia, the inheritance pattern of the disease often appears to be recessive or sporadic.

The dramatic intergenerational shifts in repeat length and en masse deletions in sperm likely underlie the reduced paternal penetrance associated with SCA8. In addition, the size of the CTA repeat tract and the interruptions within the CTG repeat on expanded SCA8 alleles may affect the overall repeat length needed to cause disease and may account for interfamilial variations in pathogenic thresholds (2,47,15,16). The remarkable instability at the SCA8 locus should provide additional examples of the variety of DNA structures and mechanisms that can be involved in human trinucleotide repeat instability.

MATERIALS AND METHODS

We identified, obtained informed consent from and collected blood samples from ataxia families. Normal controls included DNA from the spouses of ataxia patients, CEPH grandparents and anonymous DNA samples from patients being tested for blood clotting abnormalities at the University of Minnesota Molecular Diagnostic Laboratory. Separate IRB approval was obtained to use the anonymous University of Minnesota DNA samples.

Analysis of SCA8 alleles

PCR amplification was performed as described (2). Amplified products were pooled and cloned into pBluescript as described (2); at least two independent clones from each PCR product were sequenced. If sequence analysis of the clones was not concordant, presumably due to polymerase errors, then additional independent clones were sequenced. Interruption patterns observed among the ataxia families were verified for sequence accuracy by sequencing multiple family members.

Semen samples were collected from men with and without expanded SCA8 alleles. Sperm DNA isolation was performed using the Gentra Puregene DNA isolation kit (Gentra, Plymouth, MN), following the protocol for a small cell number.

Southern blot analysis

Aliquots of 5 µg of genomic DNA were digested with EcoRI, run on a 0.7% agarose gel and Southern blotted. Blots were probed with a 441 bp PCR product immediately 3′ of the CTG repeat. The primers used to make the probe were SCA8-Southern A (5′-GAA TTC ATT CCT TGC TTA CCA-3′) and SCA8-Southern B (5′-CTG CTG CAT TTT TTA AAA ATA-3′). The PCR product was cut out of the gel and random labeled using 32P.

ACKNOWLEDGEMENTS

We thank family members for their participation, A. Durand and C. Peterson for developing the pedigrees and D. Livingston for helpful discussions. This work was supported by grants from the National Ataxia Foundation and the National Institutes of Health (PO1 NS33718) to L.R. and by V.A. Research Funds to T.B.

+

To whom correspondence should be addressed at: MMC 206, 515 Delaware Street SE, Minneapolis, MN 55455, USA. Tel: +1 612 624 0901; Fax: +1 612 626 2600; Email: laura@gene.med.umn.edu

Figure 1. SCA8 allele configurations determined by sequencing. The sequence configurations are indicated by symbols on the left and summarized in the text on the right. Alleles that were sequenced included: (A) unexpanded alleles from the general population; (B) expanded alleles from small ataxia families. Representative alleles from the ataxia families were sequenced. The interfamilial variations in the size of the 3′-most uninterrupted stretch of CTGs are shown with the various repeat lengths separated by commas. Bold italicized font indicates the size of the representative allele(s) from a given family that was sequenced. (C) Expanded alleles with ≤78 CTG repeats from the general population. (D) Expanded alleles from a single large SCA8 kindred. All of the alleles shown in (D) were sequenced.

Figure 1. SCA8 allele configurations determined by sequencing. The sequence configurations are indicated by symbols on the left and summarized in the text on the right. Alleles that were sequenced included: (A) unexpanded alleles from the general population; (B) expanded alleles from small ataxia families. Representative alleles from the ataxia families were sequenced. The interfamilial variations in the size of the 3′-most uninterrupted stretch of CTGs are shown with the various repeat lengths separated by commas. Bold italicized font indicates the size of the representative allele(s) from a given family that was sequenced. (C) Expanded alleles with ≤78 CTG repeats from the general population. (D) Expanded alleles from a single large SCA8 kindred. All of the alleles shown in (D) were sequenced.

Figure 2. An abbreviated pedigree for the large SCA8 kindred showing the different allele configurations found within this family. The two cases in which allele configurations changed over a single generation are noted as case 1 and case 2. The CTA, CTG and CCG triplets are indicated by black, open and gray circles, respectively. The number of CTG repeats at the 3′ end of each allele is indicated by a subscript number. Filled pedigree symbols indicate individuals with ataxia. Haplotype analysis confirms that both branches of the family inherited the expanded repeat from a common, closely related founder.

Figure 2. An abbreviated pedigree for the large SCA8 kindred showing the different allele configurations found within this family. The two cases in which allele configurations changed over a single generation are noted as case 1 and case 2. The CTA, CTG and CCG triplets are indicated by black, open and gray circles, respectively. The number of CTG repeats at the 3′ end of each allele is indicated by a subscript number. Filled pedigree symbols indicate individuals with ataxia. Haplotype analysis confirms that both branches of the family inherited the expanded repeat from a common, closely related founder.

Figure 3.En masse contraction of SCA8 alleles in sperm. (A) Dramatic repeat length changes in patients 1 and 2 detected by Southern blotting. The repeat lengths of patients 1 and 2 contract from 500–800 repeats in blood (B) to ∼80–100 repeats in sperm (S), respectively. The probe used did not contain the CTG repeat. (B) Southern blots of blood and sperm DNA from patients with smaller expansions in their blood reveal the same trend of contractions of the expanded allele in sperm to repeat sizes that are less often associated with ataxia (below ∼100 repeats). Again, the equal intensities of the bands representing the normal and expanded alleles indicate that repeat contractions occurred in all or nearly all of the sperm with expanded alleles. (C) PCR analysis of SCA8 contractions in two patients from an unusual family with paternal disease transmission. Although contraction of repeats in sperm is again observed, the resulting alleles remain within the most penetrant size range.

Figure 3.En masse contraction of SCA8 alleles in sperm. (A) Dramatic repeat length changes in patients 1 and 2 detected by Southern blotting. The repeat lengths of patients 1 and 2 contract from 500–800 repeats in blood (B) to ∼80–100 repeats in sperm (S), respectively. The probe used did not contain the CTG repeat. (B) Southern blots of blood and sperm DNA from patients with smaller expansions in their blood reveal the same trend of contractions of the expanded allele in sperm to repeat sizes that are less often associated with ataxia (below ∼100 repeats). Again, the equal intensities of the bands representing the normal and expanded alleles indicate that repeat contractions occurred in all or nearly all of the sperm with expanded alleles. (C) PCR analysis of SCA8 contractions in two patients from an unusual family with paternal disease transmission. Although contraction of repeats in sperm is again observed, the resulting alleles remain within the most penetrant size range.

Figure 4. A model for the duplication of interrupting CCG repeats in SCA8. (A) Case 1 (from Fig. 2). During replication (not all strands shown) the newly synthesized strand loops out by intrastrand base pairing while replication proceeds on this same strand, resulting in a duplication of the CCG interruption. The increase in CTGs may or may not involve the same mechanism and need not occur during the same replication event. (B) Case 2. Only one repeat loop forms on the newly synthesized (top) strand. A possible mechanism for CTG repeat contraction is also shown in which a loop forms on the template strand while replication proceeds on the top strand. Again, the decrease in CTG repeat length that occurs 3′ to the duplication event may or may not involve the same mechanism and need not occur during the same replication event.

Figure 4. A model for the duplication of interrupting CCG repeats in SCA8. (A) Case 1 (from Fig. 2). During replication (not all strands shown) the newly synthesized strand loops out by intrastrand base pairing while replication proceeds on this same strand, resulting in a duplication of the CCG interruption. The increase in CTGs may or may not involve the same mechanism and need not occur during the same replication event. (B) Case 2. Only one repeat loop forms on the newly synthesized (top) strand. A possible mechanism for CTG repeat contraction is also shown in which a loop forms on the template strand while replication proceeds on the top strand. Again, the decrease in CTG repeat length that occurs 3′ to the duplication event may or may not involve the same mechanism and need not occur during the same replication event.

Table 1.

Repeat instability in sperm

Sample CTG repeat size in blood CTG repeat size in sperm Length of stable CTA  Contractions below ∼100 repeats 
1-1 ∼500a ∼80 12 Yesb 
2-1 ∼800a ∼100 n.a. Yesb 
3-1 80 ∼65 Yesb 
4-1 89 ∼75 11 Yesb 
4-2 115 ∼70 11 Yes 
5-1 116 ∼90 Yesb 
6-1 260 ∼170 Noc 
6-2 170 ∼160 Noc 
Sample CTG repeat size in blood CTG repeat size in sperm Length of stable CTA  Contractions below ∼100 repeats 
1-1 ∼500a ∼80 12 Yesb 
2-1 ∼800a ∼100 n.a. Yesb 
3-1 80 ∼65 Yesb 
4-1 89 ∼75 11 Yesb 
4-2 115 ∼70 11 Yes 
5-1 116 ∼90 Yesb 
6-1 260 ∼170 Noc 
6-2 170 ∼160 Noc 

aApproximate repeat sizes estimated by Southern analysis.

bSouthern data shown.

cConsistent with the contractions in sperm DNA that remain within the most pathogenic range (100–250 CTGs), these individuals are members of a family with unusual paternal disease transmission.

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