Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27

Genetic linkage studies indicate that germline variations in a gene or genes on chromosome Xq27-28 are implicated in prostate carcinogenesis. The linkage peak of prostate cancer overlies a region of approximately 750 kb containing five SPANX genes (SPANX-A1, -A2, -B, -C, and -D) encoding sperm proteins associated with the nucleus; their expression was also detected in a variety of cancers. SPANX genes are >95% identical and reside within large segmental duplications (SDs) with a high level of similarity, which confounds mutational analysis of this gene family by routine PCR methods. In this work, we applied transformation-associated recombination cloning (TAR) in yeast to characterize individual SPANX genes from prostate cancer patients showing linkage to Xq27-28 and unaffected controls. Analysis of genomic TAR clones revealed a dynamic nature of the replicated region of linkage. Both frequent gene deletion/duplication and homology-based sequence transfer events were identified within the region and were presumably caused by recombinational interactions between SDs harboring the SPANX genes. These interactions contribute to diversity of the SPANX coding regions in humans. We speculate that the predisposition to prostate cancer in X-linked families is an example of a genomic disease caused by a specific architecture of the SPANX gene cluster.     

Exclusion of the gastrin-releasing peptide receptor (GRPR) locus as a candidate gene for Rett syndrome

The gene for the gastrin-releasing peptide receptor (GRPR) has been mapped to a candidate region for Rett syndrome (RTT) on the short arm of the X chromosome. The recent report of a translocation that disrupted the gene in an individual with mental retardation and autistic behavior prompted us to examine GRPR as a possible locus for RTT. Genomic polymerase chain reaction amplification of exons followed by single-strand conformation analysis screening in 25 unrelated RTT-affected individuals and by direct sequencing in 12 others has failed to detect any mutation. No gross structural rearrangements were found by Southern analysis of DNA from six unrelated RTT-affected individuals. A high-frequency biallelic polymorphism caused by two single nucleotide substitutions in exon 2 was discovered. The allele frequencies were identical in the RTT population as compared to 100 normal control X chromosomes. This polymorphism will enable future evaluation of the GRPR locus as a candidate for other X-linked mental retardation or neurobehavioral syndromes. Am. J. Med. Genet. 78:173–175, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of Xq27-28 linkage in the international consortium for prostate cancer genetics (ICPCG) families

ABSTRACT: BACKGROUND: Genetic variants are likely to contribute to a portion of prostate cancer risk. Full elucidation of the genetic etiology of prostate cancer is difficult because of incomplete penetrance and genetic and phenotypic heterogeneity. Current evidence suggests that genetic linkage to prostate cancer has been found on several chromosomes including the X; however, identification of causative genes has been elusive. METHODS: Parametric and non-parametric linkage analyses were performed using 26 microsatellite markers in each of 11 groups of multiple-case prostate cancer families from the International Consortium for Prostate Cancer Genetics (ICPCG). Meta-analyses of the resultant familyspecific linkage statistics across the entire 1,323 families and in several predefined subsets were then performed. RESULTS: Meta-analyses of linkage statistics resulted in a maximum parametric heterogeneity lod score (HLOD) of 1.28, and an allele-sharing lod score (LOD) of 2.0 in favor of linkage to Xq27- q28 at 138 cM. In subset analyses, families with average age at onset less than 65 years exhibited a maximum HLOD of 1.8 (at 138 cM) versus a maximum regional HLOD of only 0.32 in families with average age at onset of 65 years or older. Surprisingly, the subset of families with only 2-3 affected men and some evidence of male-to-male transmission of prostate cancer gave the strongest evidence of linkage to the region (HLOD = 3.24, 134 cM). For this subset, the HLOD was slightly increased (HLOD = 3.47 at 134 cM) when families used in the original published report of linkage to Xq27-28 were excluded. CONCLUSIONS: Although there was not strong support for linkage to the Xq27-28 region in the complete set of families, the subset of families with earlier age at onset exhibited more evidence of linkage than families with later onset of disease. A subset of families with 2-3 affected individuals and with some evidence of male to male disease transmission showed stronger linkage signals. Our results suggest that the genetic basis for prostate cancer in our families is much more complex than a single susceptibility locus on the X chromosome, and that future explorations of the Xq27-28 region should focus on the subset of families identified here with the strongest evidence of linkage to this region.     

Exclusion of the familial Mediterranean fever locus as a susceptibility region for autosomal dominant familial Hibernian fever.

Autosomal dominant periodic fevers constitute a range of syndromes characterised by recurrent attacks of fever and abdominal pain. Familial Hibernian fever (FHF) has been described in only one United Kingdom based family, but two other Irish families have been found with similar clinical features. FHF resembles familial Mediterranean fever (FMF) in several clinical features, but the mode of inheritance of FHF is dominant whereas FMF is recessive. We have investigated whether autosomal dominant periodic fevers, in particular FHF, map to the FMF susceptibility locus (MEFV) on chromosome 16p13.3. We have used informative microsatellite markers flanking this locus to genotype members of the three families mentioned above. Two point and multipoint lod scores definitively excluded linkage to MEFV in the two larger families. A haplotype study confirmed these findings, indicating that FHF is genotypically as well as phenotypically distinct from FMF.     

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at θ = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23–p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene. Am. J. Med. Genet. 71:292–297, 1997. © 1997 Wiley-Liss, Inc.     

Exclusion of Kif1c as a candidate gene for anthrax toxin susceptibility

Different strains of mice possess varying degrees of susceptibility to anthrax lethal toxin (LT). Previous studies have suggested a responsible locus Ltxs1 that contains 10 or more known genes, but functional relevance has been reported for two genes, Kif1c and Nalp1b. In this study, we attempted to determine the involvement of Kif1c in anthrax susceptibility using Kif1c knockout mice. We established Kif1c knockout mice with LT-sensitive 129/Sv-derived embryonic stem cells followed by 13 backcrosses with LT-resistant C57BL/6J mice (B6) to be congenic. These knockout mice and their primary macrophages showed significantly higher sensitivity to LT than wild-type B6. However, when we replaced the remaining 129/Sv genome adjacent to the targeted Kif1c locus with the B6 genome, this sensitivity was lost. This suggested that the sensitivity to LT in the originally established Kif1c knockout mice was not due to the loss of the Kif1c gene, but was because of the presence of the 129/Sv-derived genes adjacent to the disrupted Kif1c locus. Thus, Kif1c was excluded as a candidate anthrax susceptibility gene.     

Fine mapping of the human SCIDX1 locus at Xq12-13.1

Previous linkage analysis of families with X-linked severe combinedimmunodeficiency (SCIDX1) mapped this locus to a large regionencompassing about 10 to 20 cM at Xq12–21. We have analyzedin SCIDX1 families the segregation of 7 highly polymorphic microsatellitesrepeats localized to this region, including a new polymorphicmicrosatellite at the DXS135 locus described in this study,to refine the mapping of this disease locus. The observationsof genetic recombinants within the previously defined SCIDX1-regionallow us to establish new flanking markers at the DXS135 andDXS227 loci, which significantly reduce the region harboringthe SCIDX1 locus to a distance estimated between 3 to 5 cM.The existence of multiple, highly polymorphic markers in therefined SCIDX1 region will greatly improve the accuracy of carrierdetection and prenatal diagnosis for SCIDX1.     

Linkage analysis in Rett syndrome families suggests that there may be a critical region at Xq28.

A whole X chromosome study of families in which Rett syndrome had been diagnosed in more than one member indicated that the region between Xq27 and Xqter was the most likely region to harbour a gene which may be involved in the aetiology of the disease. Further, more detailed studies of Xq28 detected weak linkage and a higher than expected sharing of maternally inherited alleles. It is suggested that there may be more than one gene involved in the aetiology of this syndrome, particularly as the very rare families in which more than one girl is affected often show variable clinical symptoms.     

L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer

To find reliable biomarkers for high-grade malignancy, the relationship between immunohistochemical L-type amino-acid transporter 1 (LAT1) expression of biopsy samples, determined with the newly developed monoclonal antibody against human LAT1, and prognosis of patients with prostate cancer, was investigated. The intensity and score of immunohistochemical LAT1 expression of first biopsy samples were assessed using the modified Sinicrope et al. method and were found to be correlated with poor survival for the study group of 114 surgically treated patients as a whole ( P  = 0.0002 and 0.0270, respectively). LAT1 intensity further had a significant relationship ( P  = 0.0057) with prognosis in pathological T3 + T4 groups. Multivariate analysis indicated that the LAT1 intensity and score were more reliable prognostic markers, compared with the Gleason score and the Ki-67 labeling index. A relationship of the LAT1 intensity and score with prognosis could also be confirmed in 63 patients with inoperable cancer ( P  = 0.0070 and <0.0001, respectively). Similarly, significant differences in prognosis were confirmed in clinical T3 + T4 groups ( P  = 0.0091 and 0.0244, respectively). Moreover, the combination of LAT1 expression and Gleason score was found to have a more reliable correlation with prognosis. Thus, elevated LAT1 expression in prostate cancers is a novel independent biomarker of high-grade malignancy, which can be utilized together with the Gleason score, which is mainly dependent on cellular and structural atypia, to assess prognosis.     

A homozygous 237-kb deletion at 1p31 identified as the locus for midline cleft of the upper and lower lip in a consanguineous family

Orofacial clefts are congenital defects that vary widely in type and severity, and can occur in isolation or in association with a variety of other defects. Herein, we describe a consanguineous family afflicted with a unique form of orofacial clefting manifesting as a facial midline defect that also involves mandibular and maxillary structures. All four affected sibs had median clefts of the upper and lower lips, tooth misalignment, and poor oral hygiene. Linkage analysis of 17 family members identified a 15.3-Mb pair recessive locus at 1p31 with a LOD score of 3.63. To the best of our knowledge, this is, to date, the first locus reported for facial midline clefting and the first recessive locus for an isolated orofacial defect. The locus harboured a novel intergenic deletion of 273 164 bp, for which all fully affected sibs were homozygous. We did not note any potentially pathogenic gene variant at the 1p31 locus via exome-sequencing analysis. The identified deletion could be harbouring a regulatory element for the gene associated with the orofacial defect. The best candidate for the putative target gene is LHX8, located 49 149 bp upstream of the deletion. The gene is known to be associated with facial development in several animals. Four other family members had a subclinical phenotype – a simple notch in the lower lip or an increase in the interdental distance between the lower incisors – indicative of very low-level expression of the trait.     

Exclusion of COL2A1 as a candidate gene in a family with Wagner-Stickler syndrome.

A large family with Wagner's vitreoretinal degeneration but none of the non-ocular features of Stickler's syndrome has been studied with gene probes for type II collagen. Recombination has been observed, thus excluding type II collagen as the site of mutation in this family. This report supports other published evidence that the Wagner-Stickler syndrome is genetically heterogeneous.     

Chromosome mapping of Rett syndrome: a likely candidate region on the telomere of Xq.

Rett syndrome (RS) is a disease of neurological development. First reported 30 years ago in 1966, its biological and genetic basis remains obscure. RS is commonly thought of as an X linked dominant disorder lethal to hemizygous males. The few familial cases would arise through mosaicism or because of occasional females failing to manifest the disorder through skewed X inactivation in relevant cell types. We have one family where the mother and daughter are affected with RS, and which can be explained according to this hypothesis. If the alternative proposal of Thomas (1996) is correct, that the lack of males affected by such disorders is the result of a high male to female ratio of germline mutations rather than of gestational lethality, then the RS gene should be located on the grandpaternal chromosome. Genomic screening with markers covering the whole X chromosome has been performed. Studies using multiple informative markers indicate that the RS locus is likely to be located close to one of the X chromosome telomeres. Further investigations in eight additional families suggest the most likely region for the RS gene to be is the distal part of Xq (Xq28).     

Mutation detection in the duplicated region of the polycystic kidney disease 1 (PKD1) gene in PKD1-linked Australian families

Screening for disease-causing mutations in the duplicated region of the PKD1 gene was performed in 17 unrelated Australian individuals with PKD1-linked autosomal dominant polycystic kidney disease. Exons 2-21 and 23-34 were assayed using PKD1-specific PCR amplification and direct sequencing. We have identified 12 novel probably pathogenic DNA variants, including five truncating mutations (Q563X, c.5105delAT, c.5159delG, S2269X, c.9847delC), two in-frame deletions (c.7472del3, c.9292del39), and two splice-site mutations (IVS14+1G>C, IVS16+1G>T). Three of the mutations (G381C, Y2185D, G2785D) were predicted to lead to the replacement of conserved amino acid residues, with ensuing changes in protein conformation. Defects in the duplicated region of PKD1 thus account for 63% of our patients. Together with the previously detected mutations (Q4041X, R4227P) in the 3 region of the gene, the study has achieved an overall mutation detection rate of 74%. In addition, we have detected 31 variants (nine novel and 22 previously published) that did not segregate with the disease and were considered to be neutral polymorphisms. Three of the nine novel polymorphisms were missense mutations with a predicted effect on protein conformation, emphasizing the problems of interpretation in PKD1 mutation screening.     

Congenic mapping of the type 1 diabetes locus, Idd3, to a 780-kb region of mouse chromosome 3: identification of a candidate segment of ancestral DNA by haplotype mapping

Type 1 diabetes in the nonobese diabetic (NOD) mouse arises as a consequence of T cell-mediated destruction of the insulin-producing beta cells of the pancreas. Although little is known of the events that initiate and subsequently drive beta-cell destruction it is clear that the entire process is under complex genetic control. At present 19 loci have been mapped that influence the development of diabetes either at the level of initiation of insulitis or at the level of progression from insulitis to overt diabetes, or both. Previously, we have mapped one of these loci, Idd3, to a 0.35-cM interval on proximal mouse chromosome 3. In the present study we have narrowed the map position of this locus to an interval of 0.15 cM by a combination of novel congenic strains and an ancestral haplotype analysis approach. We have constructed a physical contig in bacterial artificial chromosome (BAC) clones across the minimal interval. Restriction mapping of the BAC contig placed the maximum size of the Idd3 interval at 780 kb between the markers D3Nds36 and D3Nds76. To refine further the Idd3 interval we developed a series of novel single nucleotide polymorphisms (SNPs) and carried out haplotype analysis on DNA from mouse strains known to carry either Idd3 susceptibility or protective alleles. This haplotype analysis identified a 145-kb segment of ancestral DNA between the microsatellite marker D3Nds6 and the SNP 81.3. One haplotype of this ancestral segment of DNA is found in mouse strains carrying an Idd3 susceptibility allele and another is found in mouse strains carrying an Idd3 protective allelle. Within the 780-kb congenically defined interval this 145-kb segment represents the most likely location for Idd3. The Il2 gene, which encodes the cytokine interleukin 2 (IL2), maps to this interval and is a strong candidate for Idd3. To investigate whether sequence variation exists in the promoter region of the Il2 gene, which might alter its expression, we sequenced the promoter region of the Il2 gene from mouse strains carrying either an Idd3 susceptibility or resistance allele. Two sequence variants were identified, neither of which fell in known regulatory elements within the Il2 promoter. In agreement with this observation steady-state Il2 mRNA levels showed no variation between susceptible and resistant mouse strains. These data suggest that the profound protection from diabetes seen in congenic mice carrying an Idd3 protective allele is unlikely to be due to differences in the level of expression of the Il2 gene. Instead, all of the current data support our hypothesis that Idd3 corresponds to amino acid variation at the amino terminus of Il2.     

Fine mapping of the X-linked split-hand/split-foot malformation (SHFM2) locus to a 5.1-Mb region on Xq26.3 and analysis of candidate genes

Split-hand/split-foot malformation (SHFM) is a genetically heterogeneous disorder, with five known loci, that causes a lack of median digital rays, syndactyly, and aplasia or hypoplasia of the phalanges, metacarpals, and metatarsals. In the only known SHFM2 family, affected males and homozygous females exhibit monodactyly or bidactyly of the hands and lobster-claw feet. This family (1) was revisited to include additional subjects and genealogical data. All 39 affected males and three females fully expressed the SHFM, while 13 carrier females examined exhibited partial expression of SHFM. We narrowed the previously linked 22-Mb genetic interval on Xq24-q26 (2), by analyzing additional family members and typing additional markers. The results define a 5.1-Mb region with a new centromeric boundary at DXS1114 and a telomeric boundary at DXS1192. We did not identify mutations in the exons and exon/intron boundaries of 19 candidate genes. These data suggest that the mutation may lie in a regulatory region of one of these candidate genes or in another gene within the SHFM2 region with unclear role in limb development.