Abstract

Rapid developments in the elucidation of simple Mendelian traits in humans, the complexity of genotype–phenotype relationships, and the growing appreciation of complex genetic traits have conspired to focus interest on the role of modifier genes in humans. This paper reviews categories of genetic modifiers and their effects and then discusses non-Mendelian inheritance patterns involving modifier genes. Although genetic models from many disease classes of human and model systems will be considered, we focus this review on the implications for the understanding of pleiotropic malformation syndromes. Genetic modifiers have so far been molecularly defined in relatively few malformation syndromes, but the rapid acknowledgement of their critical role in human development is an exciting advance in contemporary attempts to understand the relationship of phenotype and genotype.

INTRODUCTION

The use of molecular genetic analysis to interpret and to predict the phenotype in genetic diseases initially appeared promising. However, for many human diseases, including malformation syndromes, no clear relationship of phenotype to genotype has been demonstrated. Most recent advances in the molecular genetics of pleiotropic malformation syndromes have resulted from the study of disorders attributable to high-penetrance single gene alterations. However, for some phenotypes it is becoming clear that mutations in a single gene can be insufficient to cause a phenotype without sequence alterations in another gene. Further variation in phenotype or penetrance can be caused by loss or alteration of contiguous genes, transporter proteins and activator proteins, and other classes of molecules. Modifier genes (15) have become increasingly recognized as an important source of phenotypic variation that may explicate the relationship of phenotype to genotype. This review examines some of the evidence for the importance of modifier genes in development and in human malformation syndromes.

DEFINITIONS PERTAINING TO MODIFIER GENES

We follow the convention of Nadeau (3) and refer to the major effect locus as the target gene and refer to a locus that causes an alteration of the phenotypic output of that target gene a modifier. This is in contrast to the concept advanced by Weatherall (5) that breaks genes out into three categories: primary modifiers (what Nadeau refers to as target genes and others term major locus); secondary modifiers, which are genes that affect the expression of primary modifiers; and tertiary modifiers, which are genes that alter other pathologic processes not directly related to the function of the primary modifier (5). One problem with this scheme is that it collapses allelic heterogeneity (in some cases a critical source of phenotypic variation) together with cis and trans effects of other sequence alterations of the primary modifier. In our opinion, both systems are imperfect as there is no reason to preclude the existence of a phenotype where two loci interact essentially equally, which is to say that there may not be a major locus (digenic inheritance may be considered an example) and one should expect a continuous spectrum of degrees of impact of alleles at different loci in many phenotypes. Furthermore, it is becoming clear that genes and gene products interact in networks, not linear pathways, and that the range of this interaction may be large. Coding genes as tertiary modifiers in some cases may be an example of our ignorance of the effect of a gene. Therefore, it is problematic to impose hierarchical categorization schemes on a system that displays continuous variation in the degrees and qualities of gene function. Regardless of this criticism, we have to communicate about genes and gene interactions, so some reasonable nomenclature scheme must be employed, even if it does not perfectly reflect the underlying biology.

The most common effects of modifier genes can be described as additive or multiplicative, whereby the relative risk of a disease phenotype conveyed by sequence alterations in alleles at two different loci is either the sum, or the multiple, of the risks from the individual alleles, respectively (6). Epistasis refers to a genetic interaction in which an allele from one gene will mask the phenotype caused by a mutation or sequence alteration in another gene (3). Finally, genetic modifiers may reduce the penetrance, expressivity, pleiotropy and severity of a phenotype and thus have protective effects. Conversely, an increase in penetrance, expression, pleiotropy and severity may result from a susceptibility allele or genetic modifier. Models to explain the effects of modifier genes on the penetrance, expressivity and pleiotropy of clinical findings and on mechanisms of inheritance such as dominance have been developed (2,3). Modifier genes can also affect the age of disease onset or the rate of progression (3,7).

MODIFIER GENES WERE FIRST ESTABLISHED AS IMPORTANT IN METABOLIC DISEASES

The importance of modifier genes in human disease phenotypes was demonstrated in human metabolic disorders and different modifier genes can produce a variety of clinical effects. For example, allelic heterogeneity and mutations in cis can determine pancreatic sufficiency and disease severity in cystic fibrosis [OMIM 219700; (8,9)] and polymorphisms of the TGF-β1 gene and the S and Z alleles of the α1-antitrypsin gene are risk factors for pulmonary disease in patients with two mutations in the CFTR gene (9). A modifier gene at 19q13 has been correlated with meconium ileus in cystic fibrosis in both mice and humans (10,11). Modifier loci have also been used to account for discordant liver involvement in siblings with cystic fibrosis (12).

Digenic inheritance is an alternative term that has been considered to encompass triallelic inheritance. ‘Synergistic digenic’ inheritance has been defined as a requirement for mutations in two distinct genes in the same individual for a clinical phenotype, with one mutation in either gene insufficient to cause disease (4). ‘Modifier digenic’ inheritance has been described as a mutation in a gene sufficient to cause a phenotype that is subsequently modified by a second mutation in a different gene (4). We prefer to term the former as simply digenic and the latter as a classic target-modifier pair. Perhaps not surprisingly, many examples of digenic inheritance in humans to date have involved genetically heterogeneous phenotypes (for example, retinitis pigmentosa and sensorineural deafness) (Table 1). Postulated mechanisms for digenic inheritance have included alterations in gene transcription or expression (13,14), altered protein-protein interactions (15) and an impairment of functionally related genes or proteins (Table 1) (16,17). Many other examples could be cited, but we turn our attention here to malformation syndromes.

NON-MENDELIAN INHERITANCE AND MODIFIER GENES IN HUMAN MALFORMATION SYNDROMES

Bardet–Biedl syndrome [BBS: OMIM 209900 (820)] is a human malformation syndrome for which there may be complex inheritance. The syndrome comprises obesity, pigmentary retinopathy, diabetes, polydactyly, and renal and genital malformations. There is strong evidence that the disorder is inherited in an autosomal recessive pattern. There is significant genetic heterogeneity in BBS, with at least seven known loci, from which five genes have been cloned (2125). BBS is highly pleiotropic with substantial clinical variability, but no specific phenotypic manifestations or subtypes have been definitively correlated to a particular locus or gene by linkage or mutation analysis (26,27).

Although this picture appears straightforward, there are a number of intriguing and unusual features of the genetics of BBS. The history of gene discovery in BBS is that several of the less common loci were cloned first (BBS6, initially as the cause of McKusick–Kaufman syndrome in the Amish, and then as an uncommon cause of BBS and then BBS2, followed by BBS4 and BBS1). In the early study of these genes, a substantial number of heterozygous mutations were detected in patients with BBS, but the second mutant allele could not be identified (2729). The most intriguing case is a family where there are three mutant alleles at two loci. In this family, the unaffected sib has two mutations at the BBS2 locus and the affected sib has these same two BBS2 mutations and, in addition, a third mutation in an allele of BBS6 (Table 1) (28). These data were combined with several additional families where haplotype data are consistent with a model that requires three alleles for full penetrance and this phenomenon was termed triallelic inheritance. Other studies of more BBS genes and larger numbers of patients have shown no examples of families where the inheritance could be regarded as triallelic (30). This relates to the point made in the Introduction, which is that any scheme that imposes a hierarchical, discontinuous categorization onto a continuous biologic phenomenon is problematic. Yet there are valuable lessons to be learned. First, the distinction of the concepts of penetrance from expressivity is artificial, at least for malformation syndromes. Non-penetrance may be considered as much a manifestation of our limited ability to analyze traits as it is a biologic threshold. This problem was raised by the analysis of Amish families with McKusick–Kaufman syndrome, where a homozygous parent was coded as unaffected, but she and her carrier husband had four of five children affected with McKusick–Kaufman syndrome. We could consider this pseudodominant inheritance with reduced penetrance, or it could be that the mother has subtle morphologic variants that we cannot appreciate by physical examination. In the latter case, one would be tempted to term this pseudodominant inheritance with variable expressivity. Others might hypothesize triallelic inheritance. In any case, McKusick–Kaufman and Bardet–Biedl syndromes should for now be regarded as having autosomal recessive inheritance patterns with potentially widely variable expressivity. The contributions of various modifiers to this variation is intriguing and will be exciting to study.

These concepts are intriguing because they imply that modifier genes can determine phenotypic expression in malformation syndromes and it provides a potential link between classical Mendelian inheritance and the multigenic inheritance pattern observed in complex diseases (31).

CHAPERONES AS MODIFIERS OF HUMAN DISEASE

The first identified gene for BBS, MKKS, encoded a protein with the greatest similarity to a Group II chaperonin (21,22). Chaperones and chaperonin proteins are highly conserved families of heat-shock proteins (Hsps) that bind to non-native polypeptides to inhibit the aggregation of unfolded substrate and to enable correct folding or refold denatured proteins (3234). Chaperones can therefore modify phenotypic effects that result from protein instability [for example, missense mutations in inborn errors of metabolism (3538)] or from protein aggregation [for example, polyglutamine [poly(Q)] tract expansion diseases (3941)]. In the metabolic diseases, interest in the chaperones has been reflected in the development of novel therapeutic strategies in vitro to exploit the role of chaperones in protein stabilization. DNJ, a chemical chaperone, has been shown to increase the activity of the N370S mutation in fibroblasts from a patient with Gaucher disease (42). This suggests that upregulation of chaperones could be a therapeutic target for many classes of disease.

The role of chaperone proteins in the progression of neurodegenerative diseases has been the focus of extensive research. Chaperone proteins were known to suppress the cellular toxicity resulting from protein aggregation in the poly(Q) tract diseases (43,44). Recently, one possible mechanism was elucidated by the demonstration that Hsp27 protected against the cellular toxicity resulting from protein aggregation by decreasing the formation of reactive oxygen species in a cell system model of Huntington disease (HD) (41). The loss of dopaminergic neurons associated with α-synuclein inclusions was prevented by the expression of Hsp70 in a Drosophila model of Parkinson disease (45) and intra-nuclear aggregations due to the polyalanine tract expansion were reduced by human chaperone HDJ-1 in a cellular model of oculopharyngeal muscular dystrophy (46). Other modifiers of poly(Q) diseases besides chaperone proteins have also been identified. Variation in the age of onset in HD may be modified by a polymorphism (S18Y) in the ubiquitin carboxy-terminal esterase L1 (UCHL1) gene (47) and by sequence alterations in the GRIK2 gene, which may affect disease onset by altering excitatory pathways (48).

Chaperones may also modify the effects of developmental genes because of their interaction with molecules involved in signal transduction, hormone responses and cell death (49). The heat shock protein Hsp90 has been found in association with the components and regulators of the Ras/Raf1 pathway and is required for Raf1 activation in Drosophila (50). A disturbance of the equilibrium between specific chaperones and their substrates can result in a change in intercellular signaling, favoring some developmental pathways and suppressing others (49).

Further evidence of the importance of chaperones in human development is shown by mutations in the TCBE gene as a cause of Sanjad–Sakati syndrome (hypoparathyroidism, learning disabilities, growth retardation and craniofacial dysmorphism; OMIM 241410) and Kenny–Caffey syndrome (OMIM 244460) (51). The TCBE gene encodes one of several chaperone proteins critical to the folding of α-tubulin subunits and the formation of α-β-tubulin heterodimers (51).

MODIFIER GENES IN HUMAN MALFORMATIONS: HIRSCHSPRUNG DISEASE

The inheritance of Hirschsprung disease (HSCR) is complex and has been described as multifactorial in some pedigrees and Mendelian in others (52,53). Mutations in multiple genes have been identified in HSCR, among them the RET proto-oncogene, the ligand GDNF, endothelin B receptor (ENDRB) and the ligand EDN3 (52,53). Recently, HSCR was shown to be due to the interaction of three susceptibility loci (6,54). Mutations were found in the major locus ( presumed to be the RET proto-oncogene at chromosome 10q11) in only 40% of linked families (6). The data were analyzed according to both additive and multiplicative models and the fit improved significantly with a three locus (multiplicative) model (6), with the interaction of the RET gene and two, as yet unidentified, genes on chromosomes 3p21 and 19q12 necessary and sufficient for the phenotype of short-segment HSCR (6). Other genes may also influence the HSCR phenotype, and a mutation in the L1CAM gene inherited together with a common polymorphism but no mutation in the RET gene was associated with hydrocephalus and HSCR (55).

ANIMAL MODELS FOR THE ELUCIDATION OF HUMAN MODIFIER GENES

Modifier genes in human development can be studied using model systems, of which the mouse has been considered to be most relevant. There are two main experimental approaches for the elucidation of modifier genes in mice. Gene targeting is a well known approach to generate functional insufficiency or particular mutations for a gene of interest by making a ‘knock-out’ or ‘knock-in’ mouse and further studies can be directed in mice that show phenotypic variation with the same mutation in different murine strains (3,56). For example, the severity of the otocephalic phenotype due to mutations in the Otx2 gene, a murine model of the agnathia/holoprosencephaly phenotype, is strain dependent (57). By intercrossing two different strains with subsequent backcrosses and selection of offspring based on phenotype, two loci with significant modifying effects, Otmf18 and Otmf2 were mapped (57).

Experiments breeding heterozygous ‘knock-out’ mice can also aid in the identification of interacting genes and reveal novel aspects of a disease phenotype in a candidate-type approach that is not specific for the elucidation of genetic modifiers. For example, haploinsufficiency for the JAG1 gene causes Alagille syndrome (OMIM 118450) (58,59), a pleiotropic malformation syndrome comprising intrahepatic cholestasis with bile-duct hypoplasia, pulmonic stenosis, posterior embryotoxon and vertebral anomalies that is inherited in an autosomal dominant pattern (60,61). Alagille syndrome has variable expressivity in humans and no correlation of phenotype to mutational genotype has been detected (62). Mice homozygous for jag1 mutations do not survive the embryonic period, but heterozygous mice have a less severe phenotype solely comprising anterior chamber defects of the eye (63). The JAG1 gene encodes a ligand for the Notch family of receptors and mice doubly heterozygous for both Jag1 and Notch2 mutations exhibit a complex phenotype more closely related to Alagille syndrome with paucity of the intrahepatic bile ducts, head malformations and eye defects (64). This finding implies that Notch2 can act as a genetic modifier of Jag1 in mice and the close conservation of developmental pathways between mice and humans implies that the influence of Notch2, if polymorphic in humans, could modify the Alagille phenotype in humans, possibly even contributing to the observed lack of phenotype/genotype correlation in humans (64). Although it would appear straightforward that genetic variation in alleles of a receptor would modify the phenotype of the alleles of a ligand, the situation is even more interesting. It turns out that the binding of Jag1 to Notch2 is further modified by the expression of the fringe proteins, mFng and IFng, in Chinese hamster ovary (CHO) cells (65), implying a role for other modifiers of the interaction between Jag1 and Notch2 besides Notch2 sequence alterations.

CONCLUSIONS

Knowledge of the molecular genotype of a single locus is often insufficient for the prediction of a phenotype in many human pleiotropic malformation syndromes that are commonly considered to be inherited in simple Mendelian patterns. Mutations in two genes, not necessarily by themselves disease-causing, have been shown to be necessary for some diseases, and in others, may be insufficient to cause a clinically recognizable condition unless a third sequence alteration is present. Similarly, a mutation or polymorphism in a second gene can influence a pre-existing phenotype caused by a mutation in a different gene. Although the importance of modifier genes was first recognized in humans for metabolic diseases, digenic inheritance and modifier genes have subsequently been demonstrated for human malformations and in malformation syndromes in addition to multifactorial diseases. It is likely that mutation analysis in multiple genes will become frequent in future years and may prove to have greater accuracy for phenotypic predictions. Our understanding of these clinical phenotypic issues will evolve along with the molecular dissection of development genetic networks and the elucidation of modifiers of gene expression and other perturbations of the genetic and developmental regulatory processes. The role of ligand–receptor interactions, intra- and inter-cellular signaling, chaperones and the protein degradation and apoptotic pathways, and many other processes are all potential mediators of phenotypic modification in its many guises. Although we are currently limited by our terminology and proclivity to categorize continuous biologic variation, appreciation of the molecular basis of the infinite variation of phenotypes will be a fertile research area for decades to come.

*

To whom correspondence should be addressed. Tel: +1 4155141783; Fax: +1 4154769976; Email: slavotia@peds.ucsf.edu

Table 1.

Digenic inheritance in human malformation syndromes

Phenotype Gene 1 Gene 2 Mechanism of modification Reference 
Retinitis pigmentosa ROM1+/G80insG RDS+/L185P Impaired tetramer assembly 66 
Retinitis pigmentosa ROM1+/L114insG RDS+/L185P As above 66 
Usher syndrome, type 1 USH3−/− haplotype MYO7A+/del exon 25* Altered protein interactions 15 
Juvenile retinoschisis RS ND Unknown 67 
Juvenile glaucoma MTOC+/G399V CYP1B1+/R368H Common pathway 17 
Sensorineural deafness GJB6+/deletion GJB2+/35delG Reduced GJB2 expression 14 
Sensorineural deafness GJB6+/deletion GJB2+/E47X As above 14 
Sensorineural deafness GJB6+/deletion GJB2+/167 delT As above 68 
Sensorineural deafness DFNA12 haplotype DFNA2 haplotype Additive effect 69 
Junctional epidermolysis bullosa COL17A1 R1226X/L855X LAMB3+/R635X Functionally related proteins 16 
Bardet–Biedl syndrome BBS2 Y24X/Q59X BBS6+/Q147X Unknown 28 
Bardet–Biedl syndrome BBS4 A364E/A364E BBS2 T5581/T5581 Unknown 70 
Hirschprung disease RET+/ EDNRB+/S305N Unknown 71 
Autosomal dominant polycystic kidney disease PKD2+/L736X PKD1+/haplotype Unknown 72 
WS type 2/ocular albinism MITF+944delA TYR+/R402Q Altered transcription 13 
WS type 2/ocular albinism MITF+/944delA TYR R402Q/R402Q As above 13 
WS type 3/myelomeningocele PAX3+/deletion Unknown Unknown 73 
Holoprosencephaly TGIF+/del SHH+/P424A Unknown 4 
Holoprosencephaly TGIF+/T151A SHH+/del378–380 Unknown 4 
Holoprosencephaly ZIC2+/polyQ+ SHH+/G290N Unknown 4 
Familial Mediterranean fever MEVF−/− SAA1α/α Unknown 74 
Phenotype Gene 1 Gene 2 Mechanism of modification Reference 
Retinitis pigmentosa ROM1+/G80insG RDS+/L185P Impaired tetramer assembly 66 
Retinitis pigmentosa ROM1+/L114insG RDS+/L185P As above 66 
Usher syndrome, type 1 USH3−/− haplotype MYO7A+/del exon 25* Altered protein interactions 15 
Juvenile retinoschisis RS ND Unknown 67 
Juvenile glaucoma MTOC+/G399V CYP1B1+/R368H Common pathway 17 
Sensorineural deafness GJB6+/deletion GJB2+/35delG Reduced GJB2 expression 14 
Sensorineural deafness GJB6+/deletion GJB2+/E47X As above 14 
Sensorineural deafness GJB6+/deletion GJB2+/167 delT As above 68 
Sensorineural deafness DFNA12 haplotype DFNA2 haplotype Additive effect 69 
Junctional epidermolysis bullosa COL17A1 R1226X/L855X LAMB3+/R635X Functionally related proteins 16 
Bardet–Biedl syndrome BBS2 Y24X/Q59X BBS6+/Q147X Unknown 28 
Bardet–Biedl syndrome BBS4 A364E/A364E BBS2 T5581/T5581 Unknown 70 
Hirschprung disease RET+/ EDNRB+/S305N Unknown 71 
Autosomal dominant polycystic kidney disease PKD2+/L736X PKD1+/haplotype Unknown 72 
WS type 2/ocular albinism MITF+944delA TYR+/R402Q Altered transcription 13 
WS type 2/ocular albinism MITF+/944delA TYR R402Q/R402Q As above 13 
WS type 3/myelomeningocele PAX3+/deletion Unknown Unknown 73 
Holoprosencephaly TGIF+/del SHH+/P424A Unknown 4 
Holoprosencephaly TGIF+/T151A SHH+/del378–380 Unknown 4 
Holoprosencephaly ZIC2+/polyQ+ SHH+/G290N Unknown 4 
Familial Mediterranean fever MEVF−/− SAA1α/α Unknown 74 

WS=Waardenburg syndrome.

Taken from Ming and Muenke, 2002 (4).

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