Abstract: Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)(1-9), historically termed Pick's disease(10) Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics(1-8,12) Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q2111; the tau gene also lies within this region We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon in The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10 (ref 13) This causes more frequent usage of the 5' splice site and an increased proportion of tan transcripts that include exon 10 The increase in exon 10(+) messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17 (refs 12, 14)

Abstract: The identification of genes predisposing to familial cancer is an essential step towards understanding the molecular events underlying tumorigenesis and is critical for the clinical management of affected families. Despite a declining incidence, gastric cancer remains a major cause of cancer death worldwide, and about 10% of cases show familial clustering. The relative contributions of inherited susceptibility and environmental effects to familial gastric cancer are poorly understood because little is known of the genetic events that predispose to gastric cancer. Here we describe the identification of the gene responsible for early-onset, histologically poorly differentiated, high grade, diffuse gastric cancer in a large kindred from New Zealand (Aotearoa). Genetic linkage analysis demonstrated significant linkage to markers flanking the gene for the calcium-dependent cell-adhesion protein E-cadherin. Sequencing of the E-cadherin gene revealed a G --> T nucleotide substitution in the donor splice consensus sequence of exon 7, leading to a truncated gene product. Diminished E-cadherin expression is associated with aggressive, poorly differentiated carcinomas. Underexpression of E-cadherin is a prognostic marker of poor clinical outcome in many tumour types, and restored expression of E-cadherin in tumour models can suppress the invasiveness of epithelial tumour cells. The role of E-cadherin in gastric cancer susceptibility was confirmed by identifying inactivating mutations in other gastric cancer families. In one family, a frameshift mutation was identified in exon 15, and in a second family a premature stop codon interrupted exon 13. These results describe, to our knowledge for the first time, a molecular basis for familial gastric cancer, and confirm the important role of E-cadherin mutations in cancer.

Abstract: Myotonic dystrophy (DM) is caused by a CTG expansion in the 3' untranslated region of the DM gene. One model of DM pathogenesis suggests that RNAs from the expanded allele create a gain-of-function mutation by the inappropriate binding of proteins to the CUG repeats. Data presented here indicate that the conserved heterogeneous nuclear ribonucleoprotein, CUG-binding protein (CUG-BP), may mediate the trans-dominant effect of the RNA. CUG-BP was found to bind to the human cardiac troponin T (cTNT) pre-messenger RNA and regulate its alternative splicing. Splicing of cTNT was disrupted in DM striated muscle and in normal cells expressing transcripts that contain CUG repeats. Altered expression of genes regulated posttranscriptionally by CUG-BP therefore may contribute to DM pathogenesis.

Abstract: Alpha-2-macroglobulin (alpha-2M; encoded by the gene A2M) is a serum pan-protease inhibitor that has been implicated in Alzheimer disease (AD) based on its ability to mediate the clearance and degradation of A beta, the major component of beta-amyloid deposits Analysis of a deletion in the A2M gene at the 5' splice site of 'exon II' of the bait region (exon 18) revealed that inheritance of the deletion (A2M-2) confers increased risk for AD (Mantel-Haenzel odds ratio=356, P=0001) The sibship disequilibrium test (SDT) also revealed a significant association between A2M and AD (P=000009) These values were comparable to those obtained for the APOE-epsilon4 allele in the same sample, but in contrast to APOE-epsilon4, A2M-2 did not affect age of onset The observed association of A2M with AD did not appear to account for the previously published linkage of AD to chromosome 12, which we were unable to confirm in this sample A2M, LRP1 (encoding the alpha-2M receptor) and the genes for two other LRP ligands, APOE and APP (encoding the amyloid beta-protein precursor), have now all been genetically linked to AD, suggesting that these proteins may participate in a common neuropathogenic pathway leading to AD

Abstract: We screened 75 primary hepatocellular carcinomas for somatic mutations in the entire coding region of the beta-catenin gene. We detected somatic mutations in 14 tumors; 12 were considered to cause amino acid substitutions and 2 were interstitial deletions of 51 or 195 nucleotides of genomic DNA, corresponding to exon 3. Among the 12 point mutations, 6 occurred at potential serine/threonine phosphorylation residues of codons 33, 41, or 45. The remaining six tumors contained a mutation at codon 32 (aspartic acid) or 34 (glycine), flanking to the serine residue at codon 33. By Western blot analysis, we confirmed accumulation of beta-catenin in five tumors for which frozen tissues were available; the five included tumors in which amino acid alterations had occurred at codons 32, 34, or 45, and one with a 17-amino acid deletion. Our results suggested that accumulation of beta-catenin due to amino acid substitutions at potential serine/threonine phosphorylation residues or at their neighboring codons or interstitial deletions involving exon 3 could contribute to hepatocellular carcinogenesis.

Abstract: We report here the identification of a new human homeobox gene, PITX3, and its involvement in anterior segment mesenchymal dysgenesis (ASMD) and congenital cataracts in humans. The PITX3 gene is the human homologue of the mouse Pitx3 gene and is a member of the RIEG/PITX homeobox gene family. The protein encoded by PITX3 shows 99% amino-acid identity to the mouse protein, with 100% identity in the homeodomain and approximately 70% overall identity to other members of this family. We mapped the human PITX3 gene to 10q25 using a radiation-hybrid panel. A collection of 80 DNA samples from individuals with various eye anomalies was screened for mutations in the PITX3 gene. We identified two mutations in independent patients. A 17-bp insertion in the 3´-end of the coding sequence, resulting in a frame shift, occured in a patient with ASMD and cataracts, and a G→A substitution, changing a codon for serine into a codon for asparagine, in the 5´-end of the gene occured in a patient with congenital cataracts. Both mutations cosegregate with the disease phenotype in families, and neither were found in up to 300 control individuals studied. Further expression analysis of Pitx3in the mouse supports a unique role in early ocular development, with later expression extending to the midbrain, tongue, incisors, sternum, vertebrae and limbs. These data strongly suggest a role for PITX3 in ASMD and cataracts and provide new evidence of the contribution of the RIEG/PITX gene family to the developmental program underpinning normal eye formation.

Abstract: The BRCA2 gene was identified based on its involvement in familial breast cancer. The analysis of its sequence predicts that the gene encodes a protein with 3,418 amino acids but provides very few clues pointing to its biological function. In an attempt to address this question, specific antibodies were prepared that identified the gene product of BRCA2 as a 390-kDa nuclear protein. Furthermore, direct binding of human RAD51 to each of the four single 30-amino acid BRC repeats located at the 5′ portion of exon 11 of BRCA2 was demonstrated. Such an interaction is significant, as BRCA2 and RAD51 can be reciprocally coimmunoprecipitated by each of the individual, specific antibodies and form complexes in vivo. Inferring from the function of RAD51 in DNA repair, human pancreatic cancer cells, Capan-1, expressing truncated BRCA2 were shown to be hypersensitive to methyl methanesulfonate (MMS) treatment. Exogenous expression of wild-type BRCA2, but not BRC-deleted mutants, in Capan-1 cells confers resistance to MMS treatment. These results suggest that the interaction between the BRC repeats of BRCA2 and RAD51 is critical for cellular response to DNA damage caused by MMS.

Abstract: Beta-catenin plays essential roles in both intercellular adhesion and signal transduction. As a signaling molecule, beta-catenin supplies an activating domain to the T-cell factor/lymphoid enhancer-binding factor family of DNA-binding proteins and activates gene transcription. Posttranslational stabilization of beta-catenin, leading to elevated protein levels and constitutive gene activation, has been proposed as an important step in oncogenesis. Stabilization of beta-catenin can occur through mutation to highly conserved amino acids encoded in exon 3 of the beta-catenin gene (CTNNB1). To determine whether this pathway of malignant transformation is important in prostate cancer, we analyzed 104 prostate cancer tissue specimens, 4 prostate cancer cell lines, and 3 prostate tumor xenografts for activating mutations in exon 3 of CTNNB1. Mutations were detected in 5 of the 104 prostate cancer tissue samples. Four of the five mutations involved serine or threonine residues implicated in the degradation of beta-catenin. A fifth tumor had a mutation at codon 32, changing a highly conserved aspartic acid to a tyrosine. Mutational analysis of multiple regions from several tumor samples showed that the beta-catenin mutations were present focally and therefore may occur during tumor progression.

Abstract: p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, α, or a shorter β mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, γ (splicing out exon 11) and δ (splicing out exons 11, 12, and 13). Both γ and δ p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73γ nor p73δ interact with p53, whereas p73γ showed strong interactions with all p73 isoforms, and p73δ binds efficiently p73α and p73γ but only weakly p73β. At the functional level, p73γ is significantly less efficient in activating transcription of the p21Waf1/Cip1 promoter than p53 or p73β, whereas the effect of p73δ is intermediate and comparable to that of p73α. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21Waf1/Cip1 promoter: p73β is the most efficient in inhibiting colony formation, whereas p73γ is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.

Abstract: Beta-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator downstream of the Wnt signaling pathway. Activation of the pathway by stabilization of beta-catenin has been shown to be important in the development of colorectal carcinoma, which is mainly caused by inactivating mutations of the adenomatous polyposis coli tumor suppressor gene or by activating mutations in exon 3 of the beta-catenin gene. Here, we analyzed mutations in exon 3 of the beta-catenin gene in endometrial carcinoma cases in which loss of heterozygosity at the adenomatous polyposis coli tumor suppressor gene locus has been rarely reported. We found that 10 of 76 cases had beta-catenin gene mutations. All mutations identified were single-base missense mutations on serine/threonine residues (codons 33, 37, 41, and 45), altering the glycogen synthase kinase-3beta phosphorylation consensus motif, which participates in the degradation of beta-catenin. To determine whether these beta-catenin mutations actually led to stabilization of this protein, expression of beta-catenin was analyzed immunohistochemically, and 9 of 10 cases with the beta-catenin mutation and 20 of 66 cases without it showed accumulation of beta-catenin in the cytoplasm and/or nucleus. In total, 38% of cases showed accumulation of beta-catenin. These data indicate that stabilization of beta-catenin due to mutations in exon 3 of the beta-catenin gene and other mechanisms may have an important role in development of endometrial carcinomas.

Abstract: The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function, Homozygous mutations of WT1 can be found in similar to 15% of Wilms' tumors, Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males, A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene, Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors, Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide, Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele, In contrast to Denys-Drash syndrome, no mutant protein is produced, The splice mutation leads to an imbalance of WT1 isoforms in vivo, as detected by RT-PCR on streak gonadal tissue, Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.

Abstract: Lessa (1992) introduced intron-targeted PCR, in which a noncoding intron was amplified using primers designed from highly conserved exon sequences. Introns appear to harbour a much greater degree of genetic polymorphism within and between species than exons. On the other hand, length and nucleotide sequence of exons, and exonÐintron arrangement can be highly conserved between considerably distant animal taxa. These characteristics may allow us to design sets of primers based on exon sequences to amplify flanking intron regions. Such sets of primers might function in very distant species. This study introduces two pairs of primer sets which were designed for amplifying the 1st and 2nd introns of the S7 ribosomal protein gene in fish. These primers were applied to distant fish species in order to determine their universality, and polymorphism in the amplified fragments was investigated. The DNA sequence data of the S7 ribosomal protein gene of puffer fish (Fugu rubripes), frog (Xenopus laevis) and human were derived from Cecconi et al. (1996), Mariottini et al. (1993) and Annilo et al. (1995), respectively. Exons 1, 2 and 3 of these species were aligned to determine conserved sequence regions. Because exon 1 of humans showed very poor homology with exon 1 of other species, data from puffer fish and frog were used for aligning exon 1. By contrast, highly conserved regions among these distant species were observed in exons 2 and 3. Two sets of primers were designed from the conserved sequence regions. The primer sequences to amplify the 1st intron (RP1) were 5'-TGGCCTCTTCCTTGGCCGTC-3' (S7RPEX1F) and 5'-AACTCGTCTGGCTTTTCGCC-3' (S7RPEX2R), and those for the 2nd intron (RP2) were 5'-AGCGCCAAAATAGTGAAGCC-3' (S7RPEX2F) and 5'-GCCTTCAGGTCAGAGTTCAT-3' (S7RPEX3R). The PCR reaction mixture contained 0.2 U of Taq DNA polymerase (Perkin Elmer Cetus), 0.2 mM of each dNTP, 1 μL of the manufacturerOs supplied 10× buffer, 2 mM MgCl2, 10 pmol of each primer and 10Ð50 ng of template DNA, in a final volume of 10 μL. Amplification was carried out with an initial denaturation at 95 iC for 1 min, followed by 30 cycles of amplification (denaturation at 95 iC for 30 s, annealing at 60 iC for 1 min and extension at 72 iC for 2 min, with a final extension at 72 iC for 10 min). PCR products and those digested by endonuclease were electrophoresed on a 2.5% agarose gel (Biogel) in TBE buffer (50 mM Tris, 1 mM EDTA, and 48.5 mM boric acid). Using the standard phenolÐchloroform method, crude DNA was extracted from frozen or ethanol-preserved muscles of chum salmon (Onchorhyncus keta), tuna (Thunnus spp.) and puffer fish (Fugu rubripes), each of which belonged to a different order. Results from PCR amplifications of RP1 and RP2 are shown in Fig. 1, where amplification of a single fragment was eminent in all species. Amplified fragments of salmon, tuna and puffer fish were all different in length with respect to each other, while no length difference was observed among eight tuna species (data not shown). A battery of 4-bp cutter endonucleases was applied to PCR products of yellowfin tuna (Thunnus albacares) in order to investigate intraspecific restriction site polymorphism. P R I M E R N O T E S 1255

Abstract: The BRCA2 gene was identified based on its involvement in familial breast cancer. The analysis of its sequence predicts that the gene encodes a protein with 3,418 amino acids but provides very few clues pointing to its biological function. In an attempt to address this question, specific antibodies were prepared that identified the gene product of BRCA2 as a 390-kDa nuclear protein. Furthermore, direct binding of human RAD51 to each of the four single 30-amino acid BRC repeats located at the 5* portion of exon 11 of BRCA2 was demonstrated. Such an interaction is sig- nificant, as BRCA2 and RAD51 can be reciprocally coimmu- noprecipitated by each of the individual, specific antibodies and form complexes in vivo. Inferring from the function of RAD51 in DNA repair, human pancreatic cancer cells, Ca- pan-1, expressing truncated BRCA2 were shown to be hyper- sensitive to methyl methanesulfonate (MMS) treatment. Ex- ogenous expression of wild-type BRCA2, but not BRC-deleted mutants, in Capan-1 cells confers resistance to MMS treat- ment. These results suggest that the interaction between the BRC repeats of BRCA2 and RAD51 is critical for cellular response to DNA damage caused by MMS.

Abstract: ABSTRACT: Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3′ untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3′ untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5′ exon and intron (exon and intron 1) and an additional 3′ exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (>36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5′ regulatory region contains two CCAAT boxes but lacks a TATA box. The 5′ regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIAgene, three potential SP1 binding sites and a single γ-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C→A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2+11A/G, IVS4+44C/A, and IVS6+538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7(CA)10-11CCCCCTATA (TATC)3(TCTG)5TCCG (TCTA)6was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene,HFE, were examined and found to be normal. One hemochromatosis patient, with a normalHFEgenotype, was heterozygous for the 1303C→A mutation. Furthermore, in an examination of hemochromatosis patients with mutantHFEand normalHFEgenes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.

Abstract: Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.

Abstract: Many molecular mechanisms for neural adaptation to stress remain unknown. Expression of alternative splice variants of Slo, a gene encoding calcium- and voltage-activated potassium channels, was measured in rat adrenal chromaffin tissue from normal and hypophysectomized animals. Hypophysectomy triggered an abrupt decrease in the proportion of Slo transcripts containing a "STREX" exon. The decrease was prevented by adrenocorticotropic hormone injections. In Xenopus oocytes, STREX variants produced channels with functional properties associated with enhanced repetitive firing. Thus, the hormonal stress axis is likely to control the excitable properties of epinephrine-secreting cells by regulating alternative splicing of Slo messenger RNA.

Abstract: Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3*-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3*-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3*-most intron from pre-mRNA “marks” the 3*-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5* untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

Abstract: The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLαs, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (Gsα) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.

Abstract: Background—Little information is available on phenotype-genotype correlations in familial hypertrophic cardiomyopathy that are related to the cardiac myosin binding protein C (MYBPC3) gene. The aim of this study was to perform this type of analysis. Methods and Results—We studied 76 genetically affected subjects from nine families with seven recently identified mutations (SASint20, SDSint7, SDSint23, branch point int23, Glu542Gln, a deletion in exon 25, and a duplication/deletion in exon 33) in the MYBPC3 gene. Detailed clinical, ECG, and echocardiographic parameters were analyzed. An intergene analysis was performed by comparing the MYBPC3 group to seven mutations in the β-myosin heavy-chain gene (β-MHC) group (n=52). There was no significant phenotypic difference among the different mutations in the MYBPC3 gene. However, in the MYBPC3 group compared with the β-MHC group, (1) prognosis was significantly better (P<0.0001), and no deaths occurred before the age of 40 years; (2) the age at onset of symptoms...

Abstract: The GNAS1 gene encodes the α subunit of the G protein Gs, which couples receptor binding by several hormones to activation of adenylate cyclase. Null mutations of GNAS1 cause pseudohypoparathyroidism (PHP) type Ia, in which hormone resistance occurs in association with a characteristic osteodystrophy. The observation that PHP Ia almost always is inherited maternally has led to the suggestion that GNAS1 may be an imprinted gene. Here, we show that, although Gsα expression (directed by the promoter upstream of exon 1) is biallelic, GNAS1 is indeed imprinted in a promoter-specific fashion. We used parthenogenetic lymphocyte DNA to screen by restriction landmark genomic scanning for loci showing differential methylation between paternal and maternal alleles. This screen identified a region that was found to be methylated exclusively on a maternal allele and was located ≈35 kb upstream of GNAS1 exon 1. This region contains three novel exons that are spliced into alternative GNAS1 mRNA species, including one exon that encodes the human homologue of the large G protein XLαs. Transcription of these novel mRNAs is exclusively from the paternal allele in all tissues examined. The differential imprinting of separate protein products of GNAS1 therefore may contribute to the anomalous inheritance of PHP Ia.

Abstract: B cell precursors transiently express a pre–B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of λ5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igα and Igβ. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine λ5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for λ5/14.1. The maternal allele carried a premature stop codon in the first exon of λ5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the λ5/14.1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ5/14.1. These findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human.

Abstract: Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non-Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.

Abstract: Summary Constitutional mutations of the WT1 gene, encoding a zinc-finger transcription factor involved in renal and gonadal development, are found in most patients with Denys-Drash syndrome (DDS), or diffuse mesangial sclerosis (DMS) associated with pseudohermaphroditism and/or Wilms tumor (WT). Most mutations in DDS patients lie in exon 8 or exon 9, encoding zinc finger 2 or zinc finger 3, respectively, with a hot spot (R394W) in exon 9. We analyzed a series of 24 patients, 10 with isolated DMS (IDMS), 10 with DDS, and 4 with urogenital abnormalities and/or WT. We report WT1 heterozygous mutations in 16 patients, 4 of whom presented with IDMS. One male and two female IDMS patients with WT1 mutations underwent normal puberty. Two mutations associated with IDMS are different from those described in DDS patients. No WT1 mutations were detected in the six other IDMS patients, suggesting genetic heterogeneity of this disease. We analyzed genotype/phenotype correlations, on the basis of the constitution of a WT1 mutation database of 84 germ-line mutations, to compare the distribution and type of mutations, according to the different symptoms. This demonstrated (1) the association between mutations in exons 8 and 9 and DMS; (2) among patients with DMS, a higher frequency of exon 8 mutations among 46,XY patients with female phenotype than among 46,XY patients with sexual ambiguity or male phenotype; and (3) statistically significant evidence that mutations in exons 8 and 9 preferentially affect amino acids with different functions.