Major Gene Defect Research Articles

Combinations of Low-Frequency Genetic Variants Might Predispose to Familial Pancreatic Cancer.

Familial pancreatic cancer (FPC) is an established but rare inherited tumor syndrome that accounts for approximately 5% of pancreatic ductal adenocarcinoma (PDAC) cases. No major causative gene defect has yet been identified, but germline mutations in predisposition genes BRCA1/2, CDKN2A and PALB2 could be detected in 10–15% of analyzed families. Thus, the genetic basis of disease susceptibility in the majority of FPC families remains unknown. In an attempt to identify new candidate genes, we performed whole-genome sequencing on affected patients from 15 FPC families, without detecting BRCA1/2, CDKN2A or PALB2 mutations, using an Illumina based platform. Annotations from CADD, PolyPhen-2, SIFT, Mutation Taster and PROVEAN were used to assess the potential impact of a variant on the function of a gene. Variants that did not segregate with pancreatic disease in respective families were excluded. Potential predisposing candidate genes ATM, SUFU, DAB1, POLQ, FGFBP3, MAP3K3 and ACAD9 were identified in 7 of 15 families. All identified gene mutations segregated with pancreatic disease, but sometimes with incomplete penetrance. An analysis of up to 46 additional FPC families revealed that the identified gene mutations appeared to be unique in most cases, despite a potentially deleterious ACAD9 Ala326Thr germline variant, which occurred in 4 (8.7%) of 46 FPC families. Notably, affected PDAC patients within a family carried identical germline mutations in up to three different genes, e.g., DAB1, POLQ and FGFBP3. These results support the hypothesis that FPC is a highly heterogeneous polygenetic disease caused by low-frequency or rare variants.

HIF1A: A Putative Modifier of Hemochromatosis.

HFE-related hereditary hemochromatosis (HH) is characterized by marked phenotypic heterogeneity. Homozygosity for p.C282Y is a low penetrance genotype suggesting that the HFE-HH is a multifactorial disease resulting from a complex interaction involving a major gene defect, genetic background and environmental factors. We performed a targeted NGS-based gene panel to identify new candidate modifiers by using an extreme phenotype sampling study based on serum ferritin and iron removed/age ratio. We found an increased prevalence of the HIF1A p.Phe582Ser and p.Ala588Thr variants in patients with a severe iron and clinical phenotype. Accordingly, Huh-7 cells transfected with both variants showed significantly lower HAMP promoter activity by luciferase assay. The qRT-PCR assays showed a downregulation of hepcidin and an upregulation of the HIF1A target genes (VEGF, HMOX, FUR, TMPRSS6) in cells transfected with the HIF1A-P582S vector. We identified mutations in other genes (e.g., Serpina1) that might have some relevance in single cases in aggravating or mitigating disease manifestation. In conclusion, the present study identified HIF1A as a possible modifier of the HFE-HH phenotype cooperating with the genetic defect in downregulating hepcidin synthesis. In addition, this study highlights that an NGS-based approach could broaden our knowledge and help in characterizing the genetic complexity of HFE-HH patients with a severe phenotype expression.

An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration.

Alterations in the autophagosomal–lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal–lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

Commentary: Frailty and heterogeneity in epidemiological studies.

The paper by Aalen et al. is a timely discussion of an important topic. However, it conflates two concepts, frailty and heterogeneity, which are best kept apart. I would use the term frailty only when there is clear biological evidence that a fraction of the population is either exclusively at risk, or at vastly increased risk compared with the general population, of contracting a disease. An example of the former situation is cystic fibrosis, an autosomal recessive condition, which is caused only by inherited mutations in a single gene; an example of the latter is familial adenomatous polyposis (FAP) of the colon, a dominantly inherited condition that greatly increases the risk of colon cancer. Although there is no direct estimate of the relative hazard associated with FAP, it can be surmised to be several thousand and strongly age-dependent. I would reserve the term heterogeneity for inter-individual variation in susceptibility that is much more modest and arises from factors that do not clearly single out a sub-population at greatly increased risk of the disease. Heterogeneity of susceptibility may arise from biological factors, such as polymorphisms in metabolizing enzymes or interindividual variations in efficiency of DNA repair or cell proliferation, or from purely stochastic considerations. The role of stochasticity as a factor in heterogeneity has been under-appreciated. To their credit, Aalen et al. clearly recognize the importance of stochastic factors, and they correctly note that it is not easy to distinguish between stochasticity and chaotic behaviour arising from unstable dynamical systems. I prefer to work with stochastic models. Many toxicology experiments are conducted with highly inbred strains of animals kept under identical environmental conditions. Yet, despite identical ‘nature and nurture’, these animals do not succumb to disease at identical ages, and they show considerable heterogeneity in the number and size distribution of lesions.The same would presumably be true of humans subjected to similar experimental conditions. To summarize, there are three sources of inter-individual variation in susceptibility in human populations: first, at the most fundamental level, we have stochastic variations that are present even in genetically uniform populations living under similar or identical environmental conditions; second, we have inter-individual variations in the efficiency with which fundamental biological processes are carried out; and finally, large differences in susceptibility are determined by major gene defects (e.g. FAP) or by events occurring in embryonic life that alter populations of critical cells (perhaps exemplified by testicular carcinoma, as Aalen et al. note). Methods of analysis based on stochastic models of carcinogenesis automatically address stochastic heterogeneity if (and only if) the exact (stochastic) solutions to the models are used for data analyses. In this regard, I find the discussion of carcinogenesis models in this paper unclear. The models described in the papers by Heidenreich, Meza et al., Moolgavkar et al. and Armitage and Doll cited by Aaalen et al. were not developed to describe hazard functions in individuals, as the authors state, but rather in populations of like individuals. If the exact (stochastic) solution of these models is used in homogeneous populations, stochastic heterogeneity is immediately introduced. Let Xn-1 and Xn be random variables representing the number of pre-malignant and malignant cells, respectively, in a tissue at age t and let mn be the last mutation rate. Then, the exact stochastic solution to a carcinogenesis model is equivalent to solving the following expression for the hazard function, h(t)1⁄4 P’(t)/(1-P(t))1⁄4 mn*E[Xn-1 jXn1⁄4 0], where P(t) is the probability of a malignant cell at age t and E is the expectation. Quite often, approximate

Mutation frequency of PRKAR1B and the major familial dementia genes in a Dutch early onset dementia cohort.

Genetic factors are important in all forms of dementia, especially in early onset dementia. The frequency of major gene defects in dementia has not been investigated in the Netherlands. Furthermore, whether the recently in a FTD family identified PRKAR1B gene is associated with an Alzheimer's disease (AD) like phenotype, has not been studied. With this study, we aimed to investigate the mutation frequency of the major AD and FTD genes and the PRKAR1B gene in a well-defined Dutch cohort of patients with early onset dementia. Mutation analysis of the genes PSEN1, APP, MAPT, GRN, C9orf72 and PRKAR1B was performed on DNA of 229 patients with the clinical diagnosis AD and 74 patients with the clinical diagnosis FTD below the age of 70 years. PSEN1 and APP mutations were found in, respectively 3.5 and 0.4 % of AD patients, and none in FTD patients. C9orf72 repeat expansions were present in 0.4 % of AD and in 9.9 % of FTD patients, whereas MAPT and GRN mutations both were present in 0.4 % in AD patients, and in 1.4 % resp. 2.7 % in FTD patients. We did not find any pathogenic mutations in the PRKAR1B gene. PSEN1 mutations are the most common genetic cause in Dutch AD patients, whereas MAPT and GRN mutations were found in less than 5 percent. C9orf72 repeat expansions were the most common genetic defect in FTD patients. No pathogenic PRKAR1B mutations were found in the early onset AD and FTD patients of our study.

ITI choice for the optimal management of inhibitor patients - from a clinical and pharmacoeconomic perspective.

The development of alloantibody inhibitors against factor VIII (FVIII) represents the most significant complication of haemophilia care. Inhibitors tend to develop early in the course of treatment in about 20-30% of patients with severe haemophilia who receive on-demand or prophylactic FVIII therapy. Many factors are associated with inhibitor formation, including disease severity, major FVIII gene defects, family history and non-Caucasian race, as well as age at first treatment, intensity of early treatment, use of prophylaxis and product choice. As these latter treatment-related variables are modifiable, they provide opportunity to minimize inhibitor incidence at the clinical level. Data from the Bonn Centre in Germany have indicated an overall success rate of 78% for immune tolerance induction (ITI) therapy, with a failure rate of 15% and with some treatments either ongoing (3%) or withdrawn (4%). Similarly, data from the G-ITI study, the largest international multicentre ITI study using a single plasma-derived (pd) FVIII/von Willebrand factor (VWF) product, have demonstrated success rates (complete and partial) in primary and rescue ITI of 87% and 74%, respectively, with 85% of poor prognosis patients achieving success. Favourable clinical results based on success rates and time to tolerization continue to be reported for use of pdFVIII/VWF in ITI, with pdFVIII/VWF having a particular role in patients who require rescue ITI and those with a poor prognosis for success. Data from prospective, randomized, controlled clinical studies, such as RES.I.ST (Rescue Immune Tolerance Study), are eagerly awaited. Another factor to consider with ITI therapy is cost; preliminary data from an updated decision analytic model have provided early evidence that ITI has an economic advantage compared with on-demand or prophylactic therapy.

Changes in metabolite profiles caused by genetically determined obesity in mice.

The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite–protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-013-0590-1) contains supplementary material, which is available to authorized users.

Familial pancreatic cancer—status quo

Familial pancreatic cancer (FPC) is defined by families with at least two first-degree relatives with confirmed pancreatic ductal adenocarcinoma (PDAC) that do not fulfill the criteria of other inherited tumor syndromes with an increased risk for the development of PDAC, such as hereditary pancreatitis or hereditary breast and ovarian cancer. FPC is mostly autosomal dominant inherited and presents with a heterogeneous phenotype. Although the major gene defect has not yet been identified, some important germline mutations in the BRCA2-, PALB2-, and ATM-genes are causative in some FPC families. It is suggested by experts to include high-risk individuals in a screening program with a multidisciplinary approach under research protocol conditions. However, neither biomarkers nor reliable imaging modalities for the detection of high-grade precursor lesions are yet available. Most screening programs are currently based on endoscopic ultrasound and magnetic resonance imaging, and first data demonstrated that precursor lesions (pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm) of PDAC can be identified. Timing and extent of surgery are still a matter of debate. The present review focuses on the clinical phenotype of FPC, its histopathological characteristics, known underlying genetic changes, genetic counseling, and screening.

Inhibitors: our greatest challenge. Can we minimize the incidence?

Development of alloantibodies against infused factor VIII (FVIII) is the most significant complication of haemophilia care today. Antibodies inactivate the procoagulant activity of FVIII and inhibit patients' response to replacement therapy. As inhibitors tend to develop early in the course of FVIII treatment, the challenge is to bring patients through the critical early phase of FVIII exposure without inhibitor development as the subsequent risk is much lower. Disease severity, major FVIII gene defects, family history and non-Caucasian race are major risk factors for inhibitor development. Other variables thought to play a role in inhibitor formation include age at first treatment, intensity of early treatment, use of prophylaxis and product choice [especially recombinant vs. plasma-derived von Willebrand factor (VWF)-containing concentrates]. As these treatment-related variables are modifiable, they provide opportunity to minimize inhibitor incidence at the clinical level. At present, most data regarding inhibitor development derive from retrospective studies, registry reviews, small case series and uncontrolled studies. Findings have often been conflicting, which precludes drawing definitive conclusions. Nevertheless, some clarity is beginning to emerge. Intensity of early treatment appears to be a stronger risk factor for inhibitor development than timing of first treatment. Controlled early antigen presentation via prophylaxis looks promising, particularly in conjunction with strategies to avoid immunological danger signals, but the timing of introduction and optimal regimen are not yet known. Several reports suggest that plasma-derived VWF-containing FVIII concentrates are less immunogenic than recombinant or VWF-free plasma-derived concentrates, but this is awaiting confirmation in the ongoing prospective Survey of Inhibitors in Plasma-Product Exposed Toddlers study.

Familial pancreatic cancer—current knowledge

Familial pancreatic cancer (FPC) describes families with at least two first-degree relatives with confirmed exocrine pancreatic cancer that do not fulfil the criteria of other inherited tumour syndromes with increased risks of pancreatic cancer, such as Peutz-Jeghers syndrome, hereditary pancreatitis, and hereditary breast and ovarian cancer. The inheritance of FPC is mostly autosomal dominant and with a heterogeneous phenotype. The major gene defect is yet to be identified, although germline mutations in BRCA2, PALB2 and ATM are causative in some FPC families. Expert consensus conferences considered it appropriate to screen for pancreatic cancer in high-risk individuals using a multidisciplinary approach under research protocol conditions. However, neither biomarkers nor reliable imaging modalities for the detection of high-grade precursor lesions are yet available. Most screening programmes are currently based on findings from endoscopic ultrasonography and MRI, and data has demonstrated that precursor lesions of pancreatic cancer can be identified. No consensus exists regarding the age to initiate or stop screening and the optimal intervals for follow-up. Timing and extent of surgery as a treatment for FPC are debated. This Review focuses on the clinical phenotype of FPC, its histopathological characteristics, known underlying genetic changes and associated genetic counselling and screening.

Low Frequency of CHEK2 Mutations in Familial Pancreatic Cancer

Familial pancreatic cancer (FPC) is a rare tumour syndrome and its underlying major gene defect is still unknown. Recently, CHEK2 has been identified as multi-organ cancer susceptibility gene associated with a predisposition to breast, prostate and colon cancer. Since these cancers also are associated with some FPC families, we have analysed 35 index patients of German FPC families for CHEK2 mutations. The CHEK2 * 1100delC mutation was found in 1 (3%) of 35 FPC families. Given the low expected mutation rate of up to 1.4% for CHEK2 * 1100delC in the European population our data are suggestive for possible contribution of CHEK2 mutations to a small subset of FPC.

Mice with neuron-specific accumulation of mitochondrial DNA mutations show mood disorder-like phenotypes

There is no established genetic model of bipolar disorder or major depression, which hampers research of these mood disorders. Although mood disorders are multifactorial diseases, they are sometimes manifested by one of pleiotropic effects of a single major gene defect. We focused on chronic progressive external ophthalmoplegia (CPEO), patients with which sometimes have comorbid mood disorders. Chronic progressive external ophthalmoplegia is a mitochondrial disease, which is accompanied by accumulation of mitochondrial DNA (mtDNA) deletions caused by mutations in nuclear-encoded genes such as POLG (mtDNA polymerase). We generated transgenic mice, in which mutant POLG was expressed in a neuron-specific manner. The mice showed forebrain-specific defects of mtDNA and had altered monoaminergic functions in the brain. The mutant mice exhibited characteristic behavioral phenotypes, a distorted day-night rhythm and a robust periodic activity pattern associated with estrous cycle. These abnormal behaviors resembling mood disorder were worsened by tricyclic antidepressant treatment and improved by lithium, a mood stabilizer. We also observed antidepressant-induced mania-like behavior and long-lasting irregularity of activity in some mutant animals. Our data suggest that accumulation of mtDNA defects in brain caused mood disorder-like mental symptoms with similar treatment responses to bipolar disorder. These findings are compatible with mitochondrial dysfunction hypothesis of bipolar disorder.

Mixture distribution analysis of phenotypic markers reflectingHFEgene mutations

AbstractThe goal of this study was to determine whether statistical modeling of population data for a phenotypic marker could reflect a major locus gene defect. Identifying mutations in theHFEgene makes it possible to assess the association between transferrin saturation (TS) subpopulations andHFEmutations. Data were analyzed from 27 895 white patients who attended a health appraisal clinic and who had TS and common mutations ofHFEdetermined. Mixture distribution modeling of TS was performed, and the proportion ofHFEmutations in TS subpopulations was assessed on a probability basis. Three subpopulations of TS were identified, consistent with Hardy-Weinberg conditions for major locus effects. For men, 72% of the subpopulation with the highest mean TS hadHFEgene mutations; they were primarily homozygotes or compound heterozygotes. Seventy-three percent of the subpopulation with moderate mean TS also hadHFEgene mutations; they were predominantly simple heterozygotes. Sixty-seven percent of the subpopulation with the lowest mean TS were wild-type homozygotes. Similar results were observed for women. These results suggest that statistical modeling of population clinical laboratory test data can reveal the influence of a major locus gene defect and perhaps can be applied to other aspects of body metabolism than iron. (Blood. 2003;102:4563-4566)

Several interacting genes influence the malignant hyperthermia phenotype.

Malignant hyperthermia (MH), a potentially lethal disorder of skeletal muscle calcium homeostasis, manifests only on exposure to certain anaesthetic drugs. The mode of inheritance appears to be autosomal dominant with both locus and allelic heterogeneity having been reported. Association analysis of eight MH candidate loci in UK families has indicated that several genes influence susceptibility in individual families, rather than MH simply being a major gene defect. In support of this hypothesis, we present data on a replica analysis of an independent sample of European MH families.

Early B cell defects.

The development of B lineage cells from haematopoietic stem cells in the bone marrow to mature antibody-secreting plasma cells in peripheral lymphoid tissues is a complex and tightly regulated process. The proliferation and maturation of these cells occurs in response to numerous signals from the bone marrow microenvironment, soluble cytokines, interaction with antigen and T lymphocytes and the expression of specific gene products. Lymphocyte development, in both T and B lineages, begins with the commitment of multipotent progenitors to the lymphoid lineage. It is likely that these early stages are in part dependent upon extracellular and stromal signals, though the exact mechanisms of early lymphoid commitment are not well defined. The transcription factor Ikaros appears to play an important role in these initial stages, since mice with targeted deletions of the Ikaros gene lack T, B and natural killer cell development [1]. By contrast, the transcription factors E2A and EBF appear to be more B cell-specific, in that mice that are unable to express these genes have normal numbers of T cells and myeloid cells but few if any B lineage cells [2–4]. The earliest stages of normal B cell development are antigen-independent and occur in the bone marrow in response to stromal cell contact and cytokines. From the pre-B cell stage onwards, B cell development is dependent upon the successful assembly, and functional signalling through, pre-B and B cell receptor complexes. Central to this process is expression of the individual components of the receptor and also the molecules that transduce signals from the receptor to initiate cellular events. This is diagrammatically represented in Fig. 1. The understanding of this highly organized developmental pathway comes not only from in vitro data and the generation of transgenic mice but also from the study of naturally occurring human gene abnormalities. In this review we will outline the major gene defects that result in early B lymphocyte maturation arrest and detail the clinical manifestations and the molecular pathogenesis of these conditions. Considerable emphasis will be devoted to X-linked agammaglobulinaemia (XLA), since this is the most common abnormality of B cell development. Fig. 1 The stages of B lymphocyte development in the bone marrow and periphery are outlined. The most significant B cell developmental block most commonly seen in individuals with X-linked agammaglobulinaemia (XLA) is shown by a dashed line at the pro B cell ... X-linked agammaglobulinaemia Clinical manifestations XLA was the first human immunodeficiency described for which the clinical and laboratory findings dictated effective therapy. In 1952 Bruton reported the case of an 8-year-old boy who had suffered from recurrent infections and in whom analysis of serum by protein electrophoresis revealed no demonstrable gammaglobulin fraction [5]. The boy was treated with monthly intramuscular injections of human gamma globulin with significant clinical improvement. Although there was no family history in this initial case, subsequent cases revealed a similar clinical phenotype with an X-linked pedigree [6,7]. The phenotype of XLA is characterized in its classical form by the almost complete absence of immunoglobulin of all isotypes and the profound reduction of B lymphocytes in the peripheral cir‐culation. As a result the majority of affected boys are prone to recurrent bacterial infection from the age of approximately 4–12 months following the disappearance of maternal immunoglobulin [8]. However, in a significant number of cases (21% in a survey of 44 patients) onset of infections may occur later at 3–5 years of age [9]. The infections are usually caused by pyogenic bacteria, with Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas being the most common species [8], although mycoplasmas are a significant cause of arthritis [10,11]. The site of infection varies considerably, with sinusitis, otitis media and lower respiratory tract infections being the most common [8,9]. Resistance to viral infection remains predominantly intact except for a susceptibility to enteroviral infection which most commonly results in chronic meningoencephalitis or poliomyelitis in a significant number of cases [12]. Indeed, the major cause of mortality in the series reported by Hermaszewski & Webster [9] was as a result of enteroviral meningoencephalitis. The incidence of enteroviral disease in current populations of XLA patients appears to be less frequent, a finding which may be a result of improved clinical monitoring of patients and optimal immunoglobulin substitution therapy. The disease has always been considered to be confined to the B lymphocyte compartment; however, up to 25% of patients with XLA have profound neutropenia at the time of diagnosis [13]. This may be due to endotoxaemia following severe infections experienced by these patients, or it may be a reflection of the need for the XLA gene product in myeloid cells in situations of stress.

Characterization of the NPHP1 Locus: Mutational Mechanism Involved in Deletions in Familial Juvenile Nephronophthisis

Familial juvenile nephronophthisis is an autosomal recessive, genetically heterogeneous kidney disorder representing the most frequent inherited cause of chronic renal failure in children. A gene, NPHP1, responsible for approximately 85% of the purely renal form of nephronophthisis, has been mapped to 2q13 and characterized. The major NPHP1 gene defect is a large homozygous deletion found in approximately 80% of the patients. In this study, by large-scale genomic sequencing and pulsed-field gel electrophoresis analysis, we characterized the complex organization of the NPHP1 locus and determined the mutational mechanism that results in the large deletion observed in most patients. We showed that the deletion is 290 kb in size and that NPHP1 is flanked by two large inverted repeats of approximately 330 kb. In addition, a second sequence of 45 kb located adjacent to the proximal 330-kb repeat was shown to be directly repeated 250 kb away within the distal 330-kb repeat deleting the sequence tag site (STS) 804H10R present in the proximal copy. The patients' deletion breakpoints appear to be located within the 45-kb repeat, suggesting an unequal recombination between the two homologous copies of this smaller repeat. Moreover, we demonstrated a nonpathologic rearrangement involving the two 330-kb inverted repeats found in 11 patients and, in the homozygous state, in 2 (1.3%) control individuals. This could be explained by interchromosomal mispairing of the 330-kb inverted repeat, followed by double recombination or by a prior intrachromosomal mispairing of these repeats, leading to an inversion of the NPHP1 region, followed by an interchromosomal unequal crossover event. This complex rearrangement, as well as the common deletion found in most patients, illustrates the high level of rearrangements occurring in the centromeric region of chromosome 2.

Enhanced gustatory neural responses to sugars in the diabetic db/db mouse

Sweet taste sensitivity in a genetic model of diabetes, the db/db mouse, in which a single major gene defect leads to the expression of diabetes and obesity, was studied by examining chorda tympani nerve responses to various taste stimuli, including sugars. The chorda tympani responses to four sugars, sucrose, fructose, glucose, and maltose, in adult db/db mice showed greater relative magnitudes and lower thresholds than those in adult lean mice, but responses to other basic taste stimuli, such as NaCl, HCl, and quinine HCl, were not different in the two groups. Behavioral experiments using a two-bottle preference test demonstrated that taste preference scores for the four sugars at suprathreshold concentrations, except 1.0 M, were higher in db/db than in control mice. Infant mice of 7-9 days of age possessing the genotype db/db also exhibited greater neural responses and lower thresholds for sugars than infant control mice, whereas streptozotocin-induced adult diabetic mice possessing the genotype +/+ did not exhibit larger sugar responses. These findings suggest that the enhanced sugar sensitivities observed in db/db mice are probably determined by a single major gene, db. The db gene may act on a common factor(s) involved in the stimulus-secretion coupling in the pancreatic B cell and the taste cell of db/db mice.

Genetic and physical characterization of the early-onset Alzheimer's disease AD3 locus on chromosome 14q24.3.

Genetic linkage studies have provided significant evidence that a major gene defect, AD3, for familial early-onset Alzheimer's disease (EOAD) is located at chromosome 14q24.3, between the short tandem repeat (STR) markers D14S52 and D14S53 defining a genetic size of 22.7 cM for the AD3 candidate region. We constructed a physical map of the AD3 region using yeast artificial chromosomes (YACs) selected from both the CEPH and megaCEPH YAC libraries using the AD3 linked STR markers as well as new sequence-tagged sites (STSs) designed based on YAC terminal sequences. The YAC map is contiguous in the region between D14S258 and D14S53, a region of 8.2 cM, and has an estimated physical size of 4-8 Mb. The YAC contig map was used as a framework to localize three known genes, a pseudogene and two brain expressed sequence tags (ESTs). Linkage analysis studies in two Belgian chromosome 14 EOAD families AD/A and AD/B, identified obligate recombinants in family AD/A with D14S289 and D14S61 reducing the genetic size of the candidate AD3 region substantially. The minimal AD3 candidate region measured 6.4 cM on the genetic map and is contained within six overlapping megaCEPH YACs that covered a physical distance estimated between 2 and 6 Mb. These YACs as well as other YACs in the YAC contig map are valuable resources in gene cloning efforts or genomic sequencing experiments aiming at isolating the AD3 gene.

Characterization of deletions in the LDL receptor gene in patients with familial hypercholesterolemia in the United Kingdom.

A sample of 200 patients with a clinical diagnosis of heterozygous (189) or homozygous (11) familial hypercholesterolemia (FH) attending lipid clinics in the London area have been screened for the presence of major gene defects in the low density lipoprotein (LDL) receptor gene by Southern blotting of genomic DNA with specific probes. This study is part of a project to determine the frequency of known mutations in the LDL receptor gene in this population. A new polymorphism for the enzyme Bgl II was identified by hybridization with a probe specific for the promoter plus exon 1 of the LDL receptor gene. The observed frequency of the rare allele, characterized by a Bgl II fragment of 13 kb compared with 10 kb for the common allele, was 0.08 in this group of FH patients. Several individuals who were heterozygous for the rare allele were also heterozygous for a mutation elsewhere in the LDL receptor gene that is known to cause FH. Eight different mutations, seven deletions and one duplication, were detected in a total of nine patients, accounting for 4.5% of the mutant alleles in this group. Three of the mutations are apparently identical to deletions that have been described previously in FH patients of British or European origin, while the remaining five have not been described. Two of these were in patients of Polish and Asian Indian origin, while the other three were in patients of apparently British ancestry.

Genetics of NIDDM

This brief review discusses the current level of understanding of the role of genetic defects in the etiology of non-insulin-dependent diabetes mellitus (NIDDM) and the use of molecular-genetic methods for this study. Evidence for genetic susceptibility is strong, and defects in both insulin production and action are suspect. With restriction-fragment-length polymorphisms and genomic sequencing, various candidate loci are being evaluated. Evidence that multiple genes are involved is only circumstantial. If NIDDM is genetically heterogeneous and also influenced by environmental components, population associations and linkage analyses in families may not be as easily interpreted as for diseases involving single major gene defects.