A locus for Bowen-Conradi syndrome maps to chromosome region 12p13.3

Bowen-Conradi syndrome (BCS) is a lethal autosomal recessive disorder with an estimated incidence of 1 in 355 live births in the Hutterite population. A few cases have been reported in other populations. Here, we report the results of a genome-wide scan and fine mapping of the BCS locus in Hutterite families. By linkage and haplotype analysis the BCS locus was mapped to a 3.5 cM segment (1.9 Mbp) in chromosome region 12p13.3 bounded by F8VWF and D12S397. When genealogical relationships among the families were taken into account in the linkage analysis, the evidence for linkage was stronger and the number of potentially linked regions was reduced to one. Under the assumption that all the Hutterite patients were identical by descent for a disease-causing mutation, haplotype analysis was used to infer likely historical recombinants and thereby narrow the candidate region to a chromosomal segment shared in common by all the affected children. This study also demonstrates that BCS and cerebro-oculo-facial-skeletal syndrome (COFS) are genetically distinct.     

Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin

The RP17 locus for autosomal dominant retinitis pigmentosa has previously been mapped to chromosome 17q by linkage analysis. Two unrelated South African families are linked to this locus and the identification of key recombination events assigned the RP17 locus to a 10 cM interval on 17q22. The work reported here refines the mapping of the locus from a 10 cM to a 1 cM interval between the microsatellite markers D17S1604 and D17S948. A physical map of this interval was constructed using information from the Whitehead/MIT YAC contig WC 17.8. Sequence-tagged site (STS) content mapping of seven overlapping YACs from this contig was employed in order to build the map. A BAC library was screened to cover a gap in the YAC contig and two positive BACs were identified. Intragenic polymorphisms in the retinal fascin gene provided evidence for the exclusion of this candidate as the RP17 disease gene.     

Refinement of a locus for autosomal dominant hereditary motor and sensory neuropathy with proximal dominancy (HMSN-P) and genetic heterogeneity

Hereditary motor and sensory neuropathy with proximal dominancy (HMSN-P) is an adult-onset peripheral neurodegenerative disorder which has been reported only in the Okinawa Islands, Japan. The disease locus of "Okinawa-type" HMSN-P has been previously mapped to 3q13.1, with all affected individuals sharing an identical haplotype around the locus, suggesting that the undiscovered causative mutation in HMSN-P originated from a single founder. We have newly found two large families from the western part of Japan within which multiple members developed symptoms similar to those exhibited by HMSN-P patients from Okinawa, with no record of affinal connection between the islands. Using these pedigrees with "Kansai-type" HMSN-P, we carried out a linkage study utilizing eight microsatellite markers and identified a candidate region on 3q13.1 cosegregating with the disease (maximum two-point LOD score of 8.44 at theta=0.0) overlapping with the Okinawa-type HMSN-P locus. However, the disease haplotype shared among all affected members in these families was different from that in the Okinawa kindred, suggesting allelic heterogeneity. Such allelic variation should aid in the identification of the disease-causative gene. Moreover, the allelic heterogeneity of HMSN-P in the Japanese population suggests that HMSN-P may be more common across other ethnic groups, but classified into other disease categories.     

Paroxysmal kinesigenic choreoathetosis (PKC): confirmation of linkage to 16p11-q21, but unsuccessful detection of mutations among 157 genes at the PKC-critical region in seven PKC families

Paroxysmal kinesigenic choreoathetosis (PKC) is a paroxysmal movement disorder of unknown cause. Although the PKC-critical region (PKCCR) has been assigned to the pericentromeric region of chromosome 16 by several studies of families from various ethnic backgrounds, the causative gene has not yet been identified. In the present study, we performed linkage and haplotype analysis in four new families with PKC, as well as an intensive polymerase chain reaction (PCR) based mutation analysis in seven families for a total of 1,563 exons from 157 genes mapped around the PKCCR. Consequently, the linkage/haplotype analysis revealed that PKC was assigned to a 24-cM segment between D16S3131 and D16S408, the result confirming the previously defined PKCCR, but being unable to narrow it down. Although the mutation analysis of the 157 genes was unsuccessful at identifying any mutations that were shared by patients from the seven families, two nonsynonymous substitutions, i.e., 6186C>A in exon 3 of SCNN1G and 45842A>G in exon 29 of ITGAL, which were segregated with the disease in Families C and F, respectively, were not observed in more than 400 normal controls. Thus, one of the two genes, SCNN1G and ITGAL, could be causative for PKC, but we were not able to find any other mutations that explain the PKC phenotype.     

Evidence for a common Spinocerebellar ataxia type 7 (SCA7) founder mutation in Scandinavia

Spinocerebellar ataxia type 7 (SCA7) is a neuro-degenerative disorder characterised by progressive cerebellar ataxia and macular degeneration. SCA7 is one of the least common genetically verified autosomal dominant cerebellar ataxias (ADCAs) in the world (4.5 to 11.6%), but in Sweden and Finland SCA7 is the most commonly identified form of ADCA. In an inventory of hereditary ataxias in Scandinavia (Sweden, Norway, Denmark and Finland) we identified 15 SCA7 families, eight in Sweden and seven in Finland, while no cases of SCA7 could be found in Norway or Denmark. We examined whether the relatively high frequency of SCA7 families in Sweden and Finland was the result of a common founder effect. Only two out of 15 families could be connected genealogically. However, an extensive haplotype analysis over a 10.2 cM region surrounding the SCA7 gene locus showed that all 15 families studied shared a common haplotype over at least 1.9 cM. This strongly suggests that all Scandinavian SCA7 families originate from a common founder pre-mutation.     

Construction of cosmid contigs and high-resolution restriction mapping of the Huntington disease region of human chromosome 4

The gene responsible for Huntington disease (HD) has been localizedto a 2.2 million base pair (Mbp) region between the loci D4S10and D4S98 on the short arm of human chromosome 4. As part ofa strategy originally designed to clone the gene based on itschromosomal location, we and others previously identified overlappingyeast artificial chromosome (YAC) clones covering most of thisregion. While these YAC clones were useful for initially obtaininglong-range clone continuity, a number of features of the YACsindicated that smaller clones are generally more useful in thesubsequent steps of the positional cloning strategy. In thispaper, we use these YAC clones to generate sets of overlappingcosmid clones covering most of the HD region. We Isolated alarge number of cosmids by screening a chromosome 4-specificcosmld library with labeled DNA from a minimal overlapping setof YAC clones. These cosmid clones were further analyzed byrestriction mapping and hybridization experiments, leading tothe assembly of 185 cosmids Into eleven contigs covering morethan 1.65 Mbp and to a fine-structure restriction map of theregion. Nine of these contigs cover 90 percent of the 1.7 Mbpsubregion between loci D4S125 and D4S98 where the HD gene isnow known to lie. The detailed restriction map and the cosmidclones should facilitate the identification and localizationof cDNAs and polymorphic markers, and they provide reagentsfor large scale DNA sequencing of this region of the human genome.Our results suggest that this strategy should be generally usefulfor converting YAC clones into cosmid contigs and generatinghigh-resolution restriction maps of genomioc regions of interest.     

New insights into the genetics of X-linked dystonia-parkinsonism (XDP,DYT3)

X-linked recessive dystonia-parkinsonism is a rare movement disorder that is highly prevalent in Panay Island in the Philippines. Earlier studies identified seven different genetic alterations within a 427-kb disease locus on the X chromosome; however, the exact disease-causing variant among these is still not unequivocally determined. To further investigate the genetic cause of this disease, we sequenced all previously reported genetic alterations in 166 patients and 473 Filipino controls. Singly occurring variants in our ethnically matched controls would have allowed us to define these as polymorphisms, but none were found. Instead, we identified five patients carrying none of the disease-associated variants, and one male control carrying all of them. In parallel, we searched for novel single-nucleotide variants using next-generation sequencing. We did not identify any shared variants in coding regions of the X chromosome. However, by validating intergenic variants discovered via genome sequencing, we were able to define the boundaries of the disease-specific haplotype and narrow the disease locus to a 294-kb region that includes four known genes. Using microarray-based analyses, we ruled out the presence of disease-linked copy number variants within the implicated region. Finally, we utilized in silico analysis and detected no strong evidence of regulatory regions surrounding the disease-associated variants. In conclusion, our finding of disease-specific variants occurring in complete linkage disequilibrium raises new insights and intriguing questions about the origin of the disease haplotype, the existence of phenocopies and of reduced penetrance, and the causative genetic alteration in XDP.     

Disease-promoting and -protective genomic loci on mouse chromosomes 3 and 19 control the incidence and severity of autoimmune arthritis

Proteoglycan (PG)-induced arthritis (PGIA) is a murine model of rheumatoid arthritis. Arthritis-prone BALB/c mice are 100% susceptible, whereas the major histocompatibility complex-matched DBA/2 strain is completely resistant to PGIA. To reduce the size of the disease-suppressive loci for sequencing and to find causative genes of arthritis, we created a set of BALB/c.DBA/2-congenic/subcongenic strains carrying DBA/2 genomic intervals overlapping the entire Pgia26 locus on chromosome 3 (chr3) and Pgia23/Pgia12 loci on chr19 in the arthritis-susceptible BALB/c background. Upon immunization of these subcongenic strains and their wild-type (BALB/c) littermates, we identified a major Pgia26a sublocus on chr3 that suppressed disease onset, incidence and severity via controlling the complex trait of T-cell responses. The region was reduced to 3?Mbp (11.8?Mbp with flanking regions) in size and contained gene(s) influencing the production of a number of proinflammatory cytokines. Additionally, two independent loci (Pgia26b and Pgia26c) suppressed the clinical scores of arthritis. The Pgia23 locus (～3?Mbp in size) on chr19 reduced arthritis susceptibility and onset, and the Pgia12 locus (6?Mbp) associated with low arthritis severity. Thus, we have reached the critical sizes of arthritis-associated genomic loci on mouse chr3 and chr19, which are ready for high-throughput sequencing of genomic DNA.     

Redefining the disease locus of 16q22.1-linked autosomal dominant cerebellar ataxia

The 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA; Online Mendelian Inheritance in Man [OMIN] #117210) is one of the most common ADCAs in Japan. Previously, we had reported that the patients share a common haplotype by founder effect and that a C-to-T substitution (−16C>T) in the puratrophin-1 gene was strongly associated with the disease. However, recently, an exceptional patient without the substitution was reported, indicating that a true pathogenic mutation might be present elsewhere. In this study, we clarified the disease locus more definitely by the haplotype analysis of families showing pure cerebellar ataxia. In addition to microsatellite markers, the single nucleotide polymorphisms (SNPs) that we identified on the disease chromosome were examined to confirm the borders of the disease locus. The analysis of 64 families with the −16C>T substitution in the puratrophin-1 gene revealed one family showing an ancestral recombination event between SNP04 and SNP05 on the disease chromosome. The analysis of 22 families without identifiable genetic mutations revealed another family carrying the common haplotype centromeric to the puratrophin-1 gene, but lacking the −16C>T substitution in this gene. We concluded that the disease locus of 16q-ADCA was definitely confined to a 900-kb genomic region between the SNP04 and the −16C>T substitution in the puratrophin-1 gene in 16q22.1.     

Haplotype analysis of two recurrent genomic rearrangements in the BRCA1 gene suggests they are founder mutations for the Greek population

Pertesi M, Konstantopoulou I, Yannoukakos D. Haplotype analysis of two recurrent genomic rearrangements in the BRCA1 gene suggests they are founder mutations for the Greek population. The deletions of 4.4 and 3.2 kb identified in exons 24 and 20, respectively, are two of the four most common mutations in the BRCA1 gene in Greek breast cancer patients. They have been reported previously six and three times, respectively, in unrelated Greek families. A total of 11 more families have been identified in the present study. In order to characterize these recurrent mutations as founder mutations, it is necessary to identify the disease‐associated haplotype and prove that it is shared by all the mutation carriers, suggesting that it occurred only once in a common ancestor. Haplotype analysis was performed on 24 mutation carriers and 66 healthy individuals using 10 short tandem repeat markers located within and flanking the BRCA1 gene locus, spanning a 5.9 Mb interval. Results indicate that most of the carriers of the exon 24 deletion share a common core haplotype ‘4‐7‐6‐6‐1‐3’ between markers D17S951 and D17S1299, for a stretch of 2.9 Mb, while the common haplotype for the exon 20 deletion is ‘6‐7‐4‐2‐6‐7‐1‐3’ between markers D17S579 and D17S1299, for a stretch of 3.9 Mb. Both genomic rearrangements in BRCA1 gene are Greek founder mutations, as carriers share the same, for each mutation, disease‐associated haplotype, suggesting the presence of a distinct common ancestor for both mutations.     

Phenotypic heterogeneity in British patients with a founder mutation in the FHL1 gene

Mutations in the four-and-a-half LIM domain 1 (FHL1) gene, which encodes a 280-amino-acid protein containing four LIM domains and a single zinc-finger domain in the N-terminal region, have been associated with a broad clinical spectrum of X-linked muscle diseases encompassing a variety of different phenotypes. Patients might present with a scapuloperoneal myopathy, a myopathy with postural muscle atrophy and generalized hypertrophy, an Emery-Dreifuss muscular dystrophy, or an early onset myopathy with reducing bodies. It has been proposed that the phenotypic variability is related to the position of the mutation within the FHL1 gene. Here, we report on three British families with a heterogeneous clinical presentation segregating a single FHL1 gene mutation and haplotype, suggesting that this represents a founder mutation. The underlying FHL1 gene mutation was detected by direct sequencing and the founder effect was verified by haplotype analysis of the FHL1 gene locus. A 3-bp insertion mutation (p.Phe127_Thr128insIle) within the second LIM domain of the FHL1 gene was identified in all available affected family members of the three families. Haplotype analysis of the FHL1 region on Xq26 revealed that the families shared a common haplotype. The p.Phe127_Thr128insIle mutation in the FHL1 gene therefore appears to be a British founder mutation and FHL1 gene screening, in particular of exon 6, should therefore be indicated in British patients with a broad phenotypic spectrum of X-linked muscle diseases.     

Mapping of the formin gene and exclusion as a candidate gene for the autosomal recessive form of limb-girdle muscular dystrophy.

Limb-Girdle Muscular Dystrophy (LGMD) is a myopathy with clinical and transmission heterogeneity. The recessive form, LGMD2, has been recently mapped by linkage analysis to 15q. As an attempt to identify the gene involved in this pathology, we tested as candidate gene the LD locus, called LD for limb deformity. This gene has recently been identified and mapped to chromosome 15q13-q14. It is homologous to the murine formin gene which is localized to mouse chromosome 2. Mutations in this murine gene have been shown to cause limb deformity and kidney defect. YAC clones containing the LD gene were isolated and utilised to confirm the cytogenetic localisation. Internal DNA polymorphisms of the LD locus were analyzed in LGMD2 and CEPH families. The LD gene was mapped between the alpha cardiac actin gene and the D15S24 locus. Crossovers between the LGMD2 and the LD loci excluded the LD gene as a candidate for LGMD2.     

A novel Q378X mutation exists in the transmembrane transporter protein ABCC6 and its pseudogene: implications for mutation analysis in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an inherited disorder of the elastic tissue with characteristic progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Recently mutations in the ABCC6 gene, encoding a transmembrane transporter protein, were identified as cause of the disease. Surprisingly, sequence and RFLP analysis for exon 9 with primers corresponding to flanking intronic sequence in diseased and haplotype negative members from all of our families and in a control population revealed either a homozygous or heterozygous state for the Q378X (1132C-->T) nonsense mutation in all individuals. With the publication of the genomic structure of the PXE locus we had identified the starting point of a large genomic segmental duplication within the locus in the cytogenetic interval defined by the Cy19 and Cy185 somatic cell hybrid breakpoints on chromosome 16p13.1. By means of somatic cell hybrid mapping we located this starting point telomeric to exon 10 of ABCC6. The duplication, however, does not include exon 10, but exons 1-9. These findings suggest that one or several copies of an ABCC6 pseudogene (psiABCC6) lie within this large segmental duplication. At least one copy contains exons 1-9 and maps to the chromosomal interval defined by the Cy163 and Cy11 breakpoints. Either this copy and/or an additional copy of psiABCC6 within Cy19-Cy183 carries the Q378X mutation that masks the correct identification of this nonsense mutation as being causative in pseudoxanthoma elasticum. Long-range PCR of exon 9 starting from sequence outside the genomic replication circumvents interference from the psiABCC6 DNA sequences and demonstrates that the Q378X mutation in the ABCC6 gene is associated with PXE in some families. These findings lead us to propose that gene conversion mechanisms from psiABCC6 to ABCC6 play a functional role in mutations causing PXE.     

Presymptomatic diagnosis in families with adenomatous polyposis using highly polymorphic dinucleotide CA repeat markers flanking the APC gene.

A panel of five multiallelic and highly informative dinucleotide CA repeat markers flanking the APC gene was used for presymptomatic diagnosis of familial adenomatous polyposis coli (FAP). Marker regions were amplified by PCR. DNA fragments were separated by electrophoresis in denaturing polyacrylamide gels and visualised by ethidium bromide staining. Two or more markers were found to be informative in all nine families tested, and all 23 persons at risk could be diagnosed as affected or unaffected by the disease gene, the probability being > 99.9% in 14 cases from six families in which flanking markers were informative. We found no indication for locus heterogeneity of the disease in our sample. The polyposis phenotype and its extracolonic manifestations co-segregated with a distinct haplotype determined by the markers flanking the APC gene. In one family with no remaining living affected members, we could infer the high risk haplotype from genotyping of first degree relatives. The segregation of this haplotype is consistent with the occurrence of CHRPEs in the progeny. In a sporadic case we made use of the typical early extracolonic manifestations of the disease (osteomas, desmoids) to identify the high risk haplotype. We conclude from our experience that indirect genotyping of FAP with this particular panel of closely linked and highly polymorphic microsatellite markers is a rapid, efficient, and highly reliable method for presymptomatic diagnosis of FAP.     

Comparative association analysis reveals that corneodesmosin is more closely associated with psoriasis than HLA-Cw*0602-B*5701 in German families

HLA antigens are associated with psoriasis vulgaris across populations with different ethnic background. We have previously shown that in Caucasians this association is primarily based on the class I alleles of the extended HLA haplotype 57.1 (EH57.1/I), HLA-Cw6-HLA-B57. However, it remained unclear whether HLA-Cw6 itself or a closely linked locus predisposes to the disease. An interesting candidate for this presumed locus is corneodesmosin, which is exclusively synthesized in keratinocytes. The corneodesmosin gene locus (CDSN) is only 160 kb telomeric to HLA-C and tightly associated with psoriasis. In order to find out whether EH57.1/I or a corneodesmosin variant are the susceptibility determinants on 6p, HLA class I alleles and single-nucleotide polymorphisms (SNPs) of corneodesmosin were investigated at the sequence level and analyzed by comparative association tests. Transmission disequilibrium tests (TDT) were performed in 52 nuclear families, of which 36 were fully informative for a joint comparison of HLA and CDSN with regard to association to psoriasis. The extended TDT according to Wilson was employed to test for locus interaction. Using the HLA haplotype EH57.1/I and the CDSN haplotype formed by three intragenic variant sites at nt=619 (T), 1236 (T), and 1243 (C), we obtained the best resolution of parental transmission to index cases in the trio families. On direct comparison of the contributions of the HLA and the CDSN haplotypes, there was a markedly stronger association of the corneodesmosin TTC haplotype, which is not apparent in single locus analysis. We show furthermore that there is no higher order interaction between psoriasis, HLA, and CDSN. This lack of three-locus interaction is suggestive of two independent genetic contributions to psoriasis within the major histocompatibility complex (MHC).     

Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1).

Cerebral cavernous malformations (CCM) are congenital vascular anomalies of the brain that can cause significant neurological disabilities, including intractable seizures and hemorrhagic stroke. One locus for autosomal dominant CCM ( CCM1 ) maps to chromosome 7q21-q22. Recombination events in linked family members define a critical region of approximately 2 Mb and a shared disease haplotype associated with a presumed founder effect in families of Mexican-American descent points to a potentially smaller region of interest. Using a genomic sequence-based positional cloning strategy, we have identified KRIT1, encoding a protein that interacts with the Krev-1/rap1a tumor suppressor, as the CCM1 gene. Seven different KRIT1 mutations have been identified in 23 distinct CCM1 families. The identical mutation is present in 16 of 21 Mexican-American families analyzed, substantiating a founder effect in this population. Other Mexican-American and non-Hispanic Caucasian CCM1 kindreds harbor other KRIT1 mutations. Identification of a common Mexican-American mutation has potential clinical significance for presymptomatic diagnosis of CCM in this population. In addition, these data point to a key role for the Krev-1/rap1a signaling pathway in angiogenesis and cerebrovascular disease.     

Linkage Disequilibrium and Physical Mapping of Pas1 in Mice

By using linkage disequilibrium (LD) analysis in 21 strains of known susceptibility to lung cancer and by assembling a YAC contig, we mapped to a approximately 1.5-Mb region on distal mouse chromosome 6 the Pas1 locus, the major determinant of lung cancer predisposition in mice. Our results, on the basis of haplotype and phenetic analysis, suggest that the Pas1(s) susceptibility allele is shared by several mouse-inbred strains of independent origin, which show either high or intermediate predisposition to lung tumorigenesis. Therefore, the Pas1(s) allele is probably derived from an ancestral mouse rather than from independent mutations of the same gene. We showed the feasibility of LD in common inbred strains for the fine mapping of disease loci, and provided the biological basis and the reagents for the cloning of the Pas1 gene.     

Clouston hidrotic ectodermal dysplasia (HED): genetic homogeneity, presence of a founder effect in the French Canadian population and fine genetic mapping

HED is an autosomal dominant skin disorder that is particularly common in the French Canadian population of south-west Quebec. We previously mapped the HED gene to the pericentromeric region of chromosome 13q using linkage analysis in eight French Canadian families. In this study, we extend our genetic analysis to include a multiethnic group of 29 families with 10 polymorphic markers spanning 5.1 cM in the candidate region. Two-point linkage analysis strongly suggests absence of genetic heterogeneity in HED in four families of French, Spanish, African and Malaysian origins. Multipoint linkage analysis in all 29 families generated a peak lod score of 53.5 at D13S1835 with a 1 lod unit support interval spanning 1.8 cM. Recombination mapping placed the HED gene in a 2.4 cM region flanked by D13S1828 proximally and D13S1830 distally. We next show evidence for a strong founder effect in families of French Canadian origin thereby representing the first example of a founder disease in the south-west part of the province of Quebec. Significant association was found between HED in these families and all markers analysed (Fisher's exact test, P < 0.001). Complete allelic association was detected at D13S1828, D13S1827, D13S1835, D13S141 and D13S175 (P(excess) = 1) spanning 1.3 cM. A major haplotype including all 10 associated alleles was present on 65% of affected chromosomes. This haplotype most likely represents the founder haplotype that introduced the HED mutation into the French Canadian population. Luria-Delbrück equations and multipoint likelihood linkage disequilibrium analysis positioned the gene at the D13S1828 locus (likely range estimate: 1.75 cM) and 0.58 cM telomeric to this marker (support interval: 3.27 cM) respectively.     

Phenotypic spectrum and prevalence of INPP5E mutations in Joubert Syndrome and related disorders

Joubert syndrome and related disorders (JSRD) are clinically and genetically heterogeneous ciliopathies sharing a peculiar midbrain–hindbrain malformation known as the ‘molar tooth sign''. To date, 19 causative genes have been identified, all coding for proteins of the primary cilium. There is clinical and genetic overlap with other ciliopathies, in particular with Meckel syndrome (MKS), that is allelic to JSRD at nine distinct loci. We previously identified the INPP5E gene as causative of JSRD in seven families linked to the JBTS1 locus, yet the phenotypic spectrum and prevalence of INPP5E mutations in JSRD and MKS remain largely unknown. To address this issue, we performed INPP5E mutation analysis in 483 probands, including 408 JSRD patients representative of all clinical subgroups and 75 MKS fetuses. We identified 12 different mutations in 17 probands from 11 JSRD families, with an overall 2.7% mutation frequency among JSRD. The most common clinical presentation among mutated families (7/11, 64%) was Joubert syndrome with ocular involvement (either progressive retinopathy and/or colobomas), while the remaining cases had pure JS. Kidney, liver and skeletal involvement were not observed. None of the MKS fetuses carried INPP5E mutations, indicating that the two ciliopathies are not allelic at this locus.