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Graphical abstract, There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Introduction Membranous nephropathy (MN) is the most common cause of nephrotic syndrome (NS) in adults and is a leading cause of end-stage renal disease due to glomerulonephritis. Primary MN has a strong male predominance, accounting for approximately 65% of cases; yet, currently associated genetic loci are all located on autosomes. Previous reports of familial MN have suggested the existence of a potential X-linked susceptibility locus. Identification of such risk locus may provide clues to the etiology of MN. Methods We identified 3 families with 8 members affected by primary MN. Genotyping was performed using single-nucleotide polymorphism microarrays, and serum was sent for anti-phospholipase A2 receptor (PLA2R) antibody testing. All affected members were male and connected through the maternal line, consistent with X-linked inheritance. Genome-wide multipoint parametric linkage analysis using a model of X-linked recessive inheritance was conducted, and genetic risk scores (GRSs) based on known MN-associated variants were determined. Results Anti-PLA2R testing was negative in all affected family members. Linkage analysis revealed a significant logarithm of the odds score (3.260) on the short arm of the X chromosome at a locus of approximately 11 megabases (Mb). Haplotype reconstruction further uncovered a shared haplotype spanning 2 Mb present in all affected individuals from the 3 families. GRSs in familial MN were significantly lower than in anti-PLA2R–associated MN and were not different from controls. Conclusions Our study identifies linkage of familial membranous nephropathy to chromosome Xp11.3-11.22. Family members affected with MN have a significantly lower GRS than individuals with anti-PLA2R–associated MN, suggesting that X-linked familial MN represents a separate etiologic entity., Graphical abstract

Chicken plumage color is an important economical trait in poultry breeding, as triple-yellow indigenous broilers are preferred over western commercial broilers in the Chinese market. However, the studies on the pigmentation of plumage coloration are relatively rare at present. Here, we performed a genome-wide mapping study on an F2 intercross, whose 2 founders were one hybrid commercial line “High Quality chicken Line A” that originated from the Anak red chicken and one indigenous line “Huiyang Beard” chicken that is a classical “triple-yellow” Chinese indigenous breed. Moreover, we used an automatic colorimeter that can quantitatively assess the colorations in L∗, a∗, and b∗ values. One major quantitative trait locus (QTL) on chromosome 2 was thus identified by both genome-wide association and linkage analyses, which could explain 10 to 20% of the total phenotypic variance of the b∗ measurements of the back plumage color. Using linkage analysis, 2 additional QTL on chromosome 1 and 20 were also found to be significantly associated with the plumage coloration in this cross. With additional samples from Anak red and Huiyang Beard chickens as well as pooled resequencing data from the 2 founders of this cross, we then further narrowed down the QTL regions and identified several candidate genes, such as CABLES1, CHST11, BCL2L1, and CHD22. As the effects of QTL found in this study were substantial, quantitatively measuring the coloration rather than the descriptive measurements provides stronger statistical power for the analyses. In addition, this major QTL on chromosome 2 that was associated with feather pigmentation at the genome-wide level will facilitate the future chicken breeding for yellow plumage color. In conclusions, we mapped 3 associated QTL on chromosome 1, 2, and 20. The candidate genes identified in this study shed light in the genetic basis of yellow plumage color in chicken.

Two potential candidate species for the Mediterranean aquaculture–the common dentex (Dentex dentex) and the sharpsnout seabream (Diplodus puntazzo)-are used in the present study in order to depict their genetic architecture for the first time. We have constructed the first linkage maps for both species using SNP markers derived from ddRAD sequencing. The quality of the maps produced was verified from comparative analysis with the most studied and phylogenetically related sparid species, the gilthead sea bream (Sparus aurata). A high genetic similarity was detected, based on the high number of alignments of the two new species against the reference genome of the gilthead seabream. Furthermore, a pilot study for QTL analysis per species was performed using phenotypic measurements at the juvenile stage (approximately 2 g) and revealed putative genomic areas which affect juvenile growth performance. One of those areas was located close to a candidate gene (ANGPTL6 gene) which affects growth performance. The present study improves our knowledge on the genetic architecture of those two species by presenting not only the first linkage maps, but also by providing some indications for growth performance QTL. The results can be used as a starting point to initiate further research for the genetic improvement of these two new species.

Inter- and intra-molecular allosteric interactions underpin regulation of activity in a variety of biological macromolecules. In the voltage-gated ion channel superfamily, the conformational state of the voltage-sensing domain regulates the activity of the pore domain via such long-range allosteric interactions. Although the overall structure of these channels is conserved, allosteric interactions between voltage-sensor and pore varies quite dramatically between the members of this superfamily. Despite the progress in identifying key residues and structural interfaces involved in mediating electromechanical coupling, our understanding of the biophysical mechanisms remains limited. Emerging new structures of voltage-gated ion channels in various conformational states will provide a better three-dimensional view of the process but to conclusively establish a mechanism, we will also need to quantitate the energetic contribution of various structural elements to this process. Using rigorous unbiased metrics, we want to compare the efficiency of electromechanical coupling between various sub-families in order to gain a comprehensive understanding. Furthermore, quantitative understanding of the process will enable us to correctly parameterize computational approaches which will ultimately enable us to predict allosteric activation mechanisms from structures. In this review, we will outline the challenges and limitations of various experimental approaches to measure electromechanical coupling and highlight the best practices in the field.

To analyze genetic linkage and map purple gene ( BrPur ) controlling purple inner leaves trait of Chinese cabbage, a F 2 population was constructed by selfing a F 1 plant from homozygous purple heading line ‘14S839’ and homozygous orange heading line ‘14S162’. The phenotype investigation of head color showed that the segregation ratio of purple to non-purple individuals was consistent with the expected ratio of 3:1, which indicated that purple inner leaves trait is controlled by a single dominant gene. A total of 297 SSRs in whole genome of Chinese cabbage were tested by modified Bulked Segregant Analysis (BSA) method. Two linked markers flanking BrPur , A710 and A714, were obtained and BrPur was mapped on linkage group A07 based on the sequence of these two markers. Subsequently, two new markers flanking BrPur , CL-12 and B214-87, were developed based on two known anthocyanins-related genes, Br4CL3 and Bra004214 . Genetic mapping of all markers in the F 2 mapping population showed CL-12 and B214-87 linked to BrPur with the genetic distance of 3.1 cM and 3.5 cM, respectively.

In order to better understand cross-reactions of serogroup 33 polysaccharides and the typing sera, the structure of pneumococcal capsular polysaccharide serotype 33A was elucidated. Serotype 33A has been shown to have an identical polysaccharide backbone as that of serotype 33F, with two additional sites of O-acetylation at C5, and C6 of the 3-β-Galf residue in serotype 33A. This finding is consistent with the presence of an additional functional acetyltransferase gene (wcjE) in the cps biosynthetic locus of serotype 33A compared to 33F. The identical polysaccharide backbone with at least one common O-acetylation site (C2 of 5-β-Galf) shared by serotype 33A and 33F polysaccharides is proposed to be the epitope recognized by typing serum 33b. In addition, a 5,6-di-O-acetylated →3)-β-d-Galf5,6Ac- (1→3)-β-d-Glcp-(1→ disaccharide unit, a common structural motif present in serotypes 33A, 20, and 35A polysaccharides, is proposed to be the antigenic determinant recognized by typing serum 20b. © 2013 Published by Elsevier Ltd.

A segregating population with 410 F 2 individuals from the cross MERCIA ( Rht-B1a ) × Dwarf 123 was made to identify a new major dwarfing gene carrying by novel wheat germplasm Dwarf 123. Combination of bulk segerant analysis method was used. A total of 145 SSR markers were tested for polymorphisms among parental lines and DNA bulks of F 2 population. Out of 145 primer pairs only three markers revealed corresponding polymorphism among parental lines and F 2 DNA bulks. The marker Barc20 was close to the dwarfing gene with a genetic distance of 1.8 cM, and markers Gwm513 and Gwm495 were linked to the gene with genetic distance of 6.7 and 13 cM, respectively. Linkage analysis mapped the dwarfing gene to the long arm of chromosome 4B with the order of Barc20-dwarfing gene-Gwm513-Gwm495. The Comparision between the new gene and the known Rht-B1 alleles showed that dwarfing gene Rht-Ai123 was different from the others. The identification of the new dwarfing gene and its linked markers will greatly facilitate its utilization in wheat high yield breeding for reducing plant height.

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.

We sought to partition the genetic and environmental influences on lipoprotein subclasses and identify genomic regions that may harbor genetic variants that influence serum lipoprotein levels in a sample of Gullah-speaking African-Americans. We genotyped 5,974 SNPs in 979 subjects from 418 pedigrees and used the variance component approach to compute heritability estimates, genetic and environmental correlations, and linkage analyses for selected lipoprotein subclasses. The highest heritability estimate was observed for large VLDL particle concentration (0.56 ± 0.14). Mean LDL particle size and small LDL particle concentration (−0.94) had the strongest genetic correlation estimate. The highest logarithm of odds (LOD) score detected (3.0) was on chromosome 6p24 for small LDL particle concentration. The strongest signal, obtained with the reduced sample of diabetic individuals only, was observed on chromosome 20p13 for small LDL particle concentration. The highest bivariate linkage signal (LOD 2.4) was observed on chromosome 6p24 for mean LDL particle size and small LDL particle concentration.jlr Our results suggest a significant genetic contribution to multiple lipoprotein subclasses studied in this sample and that novel loci on chromosomes 6, 10, 16, and 20 may harbor genes contributing to small, atherogenic LDL particle concentration and large, triglyceride-rich VLDL particle concentration.

American Indians have a disproportionately high rate of kidney disease likely due to a combination of environmental and genetic factors. We performed a genome wide scan of estimated glomerular filtration rate in 3665 participants of the Strong Heart Family Study to localize genes influencing kidney disease risk factors. The participants were men and women from 13 American Indian tribes recruited from 3 centers located in Arizona, the Dakotas and Oklahoma. Multipoint variance component linkage analysis was performed for each center and on the entire cohort after controlling for center effects. Modeling strategies that incorporated age, gender and interaction terms (model 1) and another that also controlled for diabetes mellitus, systolic and diastolic blood pressure, body mass index, low density and high density lipoproteins, triglycerides and smoking status (model 2) were used. Significant evidence for linkage in the Arizona group was found on chromosome 12p12.2 at 39cM (nearest marker D12S310) using model 1. Additional loci with very suggestive evidence for linkage were detected at 1p36.31 for all groups using both models and at 2q33.3 and 9q34.2 for the Dakotas group each using model 1. No significant evidence for additive interaction with diabetes, hypertension or obesity was noted. This evidence for linkage of a quantitative trait locus influencing estimated glomerular filtration rate to a region of chromosome 12p in a large cohort of American Indians will be worth studying in more detail in the future.

Background Schizophrenia is a highly heritable and complex disorder. Multiple genes are likely to be involved, complicating genetic research into the etiology of this disorder. Intermediate phenotypes or endophenotypes may facilitate genetic research if they display a simpler mode of transmission than schizophrenia itself, i.e., if they reflect more closely the underlying genetic effects. Methods Twenty-five multigenerational families with multiple members affected with schizophrenia (180 subjects) were administered an extensive neuropsychological, psychophysiological, and personality test battery. Familial correlations were calculated to select heritable traits. Subsequent heritability analysis followed by commingling and segregation analysis were performed to unravel the pattern of transmission and to estimate heritability. Results Five traits, including sensorimotor gating, openness, verbal fluency, early visual perception, and spatial working memory, showed moderate familial correlations. Heritability estimates for these traits ranged from 37% to 54%. A major gene model resembling dominant transmission was found for both sensorimotor gating and openness. Verbal fluency, early visual perception, and spatial working memory may be accounted for by polygenic, multifactorial, or environmental effects. Conclusions Only 2 of 13 candidate endophenotypes showed a simple mode of transmission useful for successful application in molecular genetic research: sensorimotor gating and openness. To our knowledge, this is the first study to investigate the pattern of transmission for these traits.

Genetic isolates are exceptional resources for the detection of susceptibility genes for complex diseases because of the potential reduction in genetic and clinical heterogeneity. However, the outcome of these mapping efforts is dependent upon the demographic history of a given isolated population, with the most significant factors being a constant population size, the number of generations since founding, and the pathogenic loci and their allele frequencies among founders. Here we employed a cross-isolate genome-wide multipoint linkage study design using uniform genetic and clinical methods in four Daghestan ethnically and demographically diverse isolates with an aggregation of schizophrenia. Our previous population-genetics study showed that Daghestan has an extremely high genetic diversity between ethnic populations and a low genetic diversity within them. The isolates selected for this study include some with more than 200 and some with fewer than 100 generations of demographical history since their founding. Updated clinical data using DSM-IV criteria showed between-isolate differences in aggregation of distinct types of schizophrenia: one of the isolates had a predominant aggregation of disorganized schizophrenia, while the other three had predominantly paranoid schizophrenia. The summarized cross-isolate results indicated prominent within and between-isolate differences in clinical and genetic heterogeneity: the most ancient isolates have roughly twofold fewer incidences of distinct clinical phenotypes and fewer linked genomic regions compared to the demographically younger isolates, which exhibit higher clinical and genetic heterogeneity. Affected individuals in the demographically ancient isolate of ethnic Dargins (No. 6022) who suffered from disorganized schizophrenia showed the highest linkage evidence at 17p11–p12 (LOD = 3.73), while isolates with a predominant aggregation of paranoid schizophrenia (Nos. 6005, 6011, and 6034) showed the highest linkage evidence at 22q11 (LOD = 3.0 and 4.4). The unified clinical, genomic, and statistical design we used enabled us to separate the linked and unlinked pedigrees in an unbiased fashion for each genomic location. Overall maximized heterogeneity lod scores for the combined pedigrees ranging from 3.5 to 8.7 were found at 2p24, 10q26, 11q23, 12q24, 17p11–p12, 22q11, and 22q13. The cross-isolate homogeneity in linkage patterns may be ascribed to an identical-by-descent “metahaplotype” block with pathogenic loci derived from the Daghestan ethnic groups’ common ancestral metapopulation, while the cross-isolate differences may reflect differences in gene drift and recombination events in the history of local isolates. The results obtained support the notion that mapping genes of any complex disease (e.g., schizophrenia) in demographically older genetic isolates may be more time and cost effective in comparison with demographically younger isolates, especially in genetically heterogeneous outbred populations, due to higher clinical and genetic homogeneity of the primary isolates. A study at higher genotyping density across the regions of interest and fluorescence in situ hybridization analyses are currently underway.

Genetics of progressive renal failure in diabetic kidney disease Diabetic kidney disease is a microvascular complication that is observed in a minority of patients with long-standing hyperglycemia. Diabetic nephropathy (DN) is associated with shortened patient survival, severe morbidity, and increased health care costs. Unfortunately, the incidence rates of DN continue to increase in Western societies, and DN is now the most common reported cause of end-stage renal disease in developed nations. DN results from a complex interplay between inherited and environmental factors. This article reviews the data that support an inherited basis for susceptibility to DN by summarizing familial aggregation studies, genome-wide linkage, and population-based association analyses in diabetic and nondiabetic kidney disease. Recent evidence linking genes involved in the regulation of endothelial function with genetic predisposition to albuminuria is presented. The integration of carefully designed genetic linkage and association studies with gene expression experiments in human and animal models of diabetic kidney disease appear to offer great promise for detecting the molecular mechanisms underlying susceptibility to DN.

Disseminated superficial porokeratosis (DSP) is a rare autosomal dominant epidermal keratinization disorder of lesions characterized by cornoid lamella with parakeratosis, hyperkeratosis, and loss of granular layers. The genetic basis for this disease is unknown. Through a proband with a diagnosis of DSP, we identified a large four-generation Chinese family with multiple DSP-affected members from Anhui province in China. After excluding the linkage of the disease phenotype to two known loci for disseminated superficial actinic porokeratosis in this family, we performed a genome-wide linkage analysis using 387 microsatellite markers and identified a novel disease locus for DSP at 18p11.3. Our subsequent fine mapping and haplotype analyses further narrowed down the disease locus into an 18.7 cM region between the telomere and D18S391 with a maximum two-point LOD (logarithm of the odds) score of 4.82 (theta =0.00) at D18S1138. Therefore, this study provides strong linkage evidence for a DSP locus at 18p11.3.

A genome scan for diabetic nephropathy in African Americans.BackgroundThere is substantial evidence for a genetic contribution to diabetic nephropathy susceptibility in the African American population, but little is known about location or identity of susceptibility genes.MethodsDNA samples were collected from 206 type 2 diabetes (T2DM) and end-stage renal disease (ESRD)/nephropathy-affected sib pairs from 166 African American families (355 affected individuals). A genome scan was performed and data analyzed using nonparametric linkage regression (NPLR) analysis and ordered subsets analysis (OSA) methods.ResultsIn initial NPLR analyses no logarithm of odds (LOD) scores >2.0 were observed. Four loci had LOD scores ≥1.0, with LOD = 1.43 at 29 cM on chromosome 7p the highest. NPLR analyses of multilocus interactions detected 6 loci (7p, 12p, 14q, 16p, 18q, and 21q) with LOD scores 1.15 to 1.63. NPLR analyses evaluating phenotypic interactions revealed multiple locations with evidence (P < 0.05) for interactions with age-at-onset of ESRD (9 loci), duration of diabetes before onset of ESRD (19 loci), and age-at-onset of diabetes (14 loci). Several loci identified by NPLR analyses were also identified using OSA. OSA revealed evidence for a nephropathy locus at 135 cM on chromosome 3 in an estimated 29% of the families (LOD = 4.55 in the optimal subset). Additional linkage evidence, LOD = 3.59, was observed on chromosome 7p (37% of the families, longer duration of diabetes prior to diagnosis of ESRD), and 18q (max. LOD = 3.72; 64% of the families, early diabetes diagnosis). The 7p linkage has been observed in a recent genome scan of African American type 2 diabetes.ConclusionThis first genome scan of diabetic nephropathy in African Americans reveals evidence for susceptibility loci on chromosomes 3q, 7p, and 18q. The 7p locus may represent a type 2 diabetes susceptibility locus.

Familial juvenile hyperuricemic nephropathy: Detection of mutations in the uromodulin gene in five Japanese families. Background Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal-dominant disease characterized by hyperuricemia of underexcretion type, gout, and chronic renal failure. We previously reported linkage on chromosome 16p12 in a large Japanese family designated as family 1 in the present study. Recent reports on the discovery of mutations of the uromodulin ( UMOD ) gene in families with FJHN encouraged us to screen UMOD mutations in Japanese families with FJHN, including family 1. Methods Six unrelated Japanese families with FJHN were examined for mutations of the UMOD gene by direct sequencing. To confirm the results of the mutation screening, parametric linkage analyses were performed using markers in 16p12 region and around other candidate genes of FJHN. Results Five separate heterozygous mutations (Cys52Trp, Cys135Ser, Cys195Phe, Trp202Ser, and Pro236Leu) were found in five families, including family 1. All mutations were co-segregated with the disease phenotype in all families, except for family 1, in which an individual in the youngest generation was found as a phenocopy by the genetic testing. Revised multipoint linkage analysis showed that the UMOD gene was located in the interval showing logarithm of odds (LOD) score above 6.0. One family carrying no mutation in the UMOD gene showed no linkage to the medullary cystic kidney disease type 1 (MCKD1) locus, the genes of hepatocyte nuclear factor-1β (HNF-1β), or urate transporters URAT1 and hUAT. Conclusion Our results gave an evidence for the mutation of the UMOD gene in the majority of Japanese families with FJHN. Genetic heterogeneity of FJHN was also confirmed. Genetic testing is necessary for definite diagnosis in some cases especially in the young generation.

Punctate palmoplantar keratodermas (PPK) is a rare autosomal dominant cutaneous disorder characterized by numerous hyperkeratotic papules that are irregularly distributed on the palms and soles. The genetic basis for this disease is unknown. We performed a genome-wide search in two Chinese families with punctate PPK to map the chromosome location of the responsible gene. We identified a locus at chromosome 8q24.13-8q24.21 with a cumulative maximum two-point LOD score of 5.41 at markers D8S1793 and D8S1774 (at recombination fraction theta=0.00). Haplotype analysis indicated that the disease gene is located within 9.20 cM region between markers D8S1804 and D8S1720. It is the first locus identified for the punctate PPK. This study provides a map location for isolation of a disease gene-causing punctate PPK.

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant skin disorder. It is also called “reticulate acropigmentation of Dohi” or “symmetric dyschromatosis of the extremities”. The DSH locus has recently been mapped to chromosome 1q21 and pathogenic mutations were identified in the DSRAD gene encoding double-stranded RNA-specific adenosine deaminase in Japanese patients with DSH. We report here two novel point mutations, Q513X(1537C>T) and R916W(2746C>T) in the DSRAD gene identified in two Chinese families, respectively. These data suggest that mutations in DSRAD were also associated with DSH in Chinese. This is the first report on DSRAD as the causative gene of DSH in the Chinese population.

In our ongoing effort to identify genes influencing the biological pathways that underlie the metabolic disturbances associated with obesity, we performed genome-wide scanning in 2,209 individuals distributed over 507 Caucasian families to localize quantitative trait loci (QTLs), which affect variation of plasma lipids. Pedigree-based analysis using a quantitative trait variance component linkage method that localized a QTL on chromosome 7q35-q36, which linked to variation in levels of plasma triglyceride [TG, logarithm of odds (LOD) score = 3.7] and was suggestive of linkage to LDL-cholesterol (LDL-C, LOD = 2.2). Covariates of the TG linkage included waist circumference, fasting insulin, and insulin:glucose, but not body mass index or hip circumference. Plasma HDL-cholesterol (HDL-C) levels were suggestively linked to a second QTL on chromosome 12p12.3 (LOD = 2.6). Five other QTLs with lower LOD scores were identified for plasma levels of LDL-C, HDL-C, and total cholesterol. These newly identified loci likely harbor genetic elements that influence traits underlying lipid adversities associated with obesity.

Multiple familial trichoepithelioma (MFT) is an autosomal dominant skin disease characterized by the presence of many small benign tumors with pilar differentiation predominantly on the face. The first locus has been previously mapped to chromosome 9p21, but no gene for MFT has been identified to date. To identify the disease gene in a large Chinese family, we initially performed linkage analysis with microsatellite markers from 9p21, but failed to confirm the linkage to this region. Previous publications showed MFT and familial cylindromatosis (FC) can occur within one family and in a single person. Therefore, we speculated that the cylindromatosis gene (CYLDI gene) responsible for FC may be related to the pathogenesis of MFT. In view of that, we genotyped all available individuals using 11 microsatellite markers spanning the CYLDI gene region at 16q12-q13. We identified the linkage of MFT to this region. Mutation analysis in the CYLDI gene detected a frameshift mutation, designated as c.2355-2358delCAGA. The study firstly identified the cylindromatosis gene responsible for MFT and showed that different mutations of the CYLDI gene can give rise to distinct clinical and histological expression such as FC and MFT.

Freckles are numerous pigmented macules on the face commonly occurring in the Caucasian and Chinese population. As freckling is considered as an independent trait, no gene or locus for it has been identified to date. Here we performed genome-wide scan for linkage analysis in a multigeneration Chinese family with freckles. A maximum LOD score of 4.26 at a recombination fraction of 0 has been obtained with marker D4S1566. Haplotype analysis localized the freckles locus to a 16 Mbp region flanked by D4S2952 and D4S1607. We have thus mapped the gene for freckles to chromosome 4q32-q34. This will aid future identification of the responsible gene, which will be very useful for the understanding of the molecular mechanism of freckles.

25 páginas, 1 figura, 1 tabla. Fourth International Research Workshop on Alopecia Areata. Washington, 31 oct.- 2 nov. 2002., Alopecia areata affects approximately 4.6 million individuals in the United States alone. It is typified by patchy hair loss on the scalp that can progress to cover the entire scalp (alopecia totalis) and eventually the entire body (alopecia universalis). Despite the high incidence of this condition, its genetic basis is largely unknown. It is now generally accepted, however, that it fits the paradigm of a complex trait, in which a combination of genetic and environmental factors results in the final phenotype. Genetic studies have been limited thus far to association analyses, which suggest that a permissive HLA status may potentiate the development of alopecia areata. A systematic screen for identifying the primary genetic mechanisms underlying this disorder has never before been undertaken, however. Here we discuss our approach to the identification of susceptibility genes for alopecia areata. In particular, we recently initiated a comprehensive genetic analysis by performing a genome-wide scan in a collection of alopecia families with multiple affected family members. There are currently a number of examples of complex diseases of the skin, such as psoriasis and atopic dermatitis, in which genetic studies are being undertaken that substantiate the timeliness of this approach. We anticipate that these studies will lead to the identification of the susceptibility genes and provide a foundation for understanding how they interact with each other and with other variables, such as the immune system and environmental factors., This study was supported in part by grants from the National Alopecia Areata Foundation (AMC and AZ), by the General Administrator, Israel (AZ), and NIH grants N01-AR-0–2249 (NIH AA Registry), K01-HG00055 (DG), MH59492 (JO), and HG00008 (JO).

Objective : The aims of the present study were: (1) to evaluate the extent and mode of inheritance of hand osteoarthritis by using a large sample of ethnically homogeneous pedigrees of Caucasian origin; (2) to examine whether the synthetic measure of osteoarthritis according to Kellgren and Lawrence (K–L) and the more specific measure, namely, the extent of osteophytes development, have a similar putative genetic determination and pattern of biological inheritance and (3) to test the hypothesis that hand osteoarthritis dependent phenotypes are linked to the 11q 12-13 chromosomal region. Methods : The population of the present study comprised 1190 Chuvashians (Russian Federation) belonging to 295 nuclear families. Segregation analysis was carried out on a total sample. Sub-sample of 571 individuals was used to conduct Transmission/disequilibrium test (TDT) and model-based linkage analysis. Results : Adjusted for age, sex and other covariates, both OA phenotypes showed significant familial aggregation. The model fitting analysis strongly supported the hypothesis of a major gene effect on study traits. The inferred major gene explained about 52% of the osteophyte score (OPS) and 49% of the K–L score variation adjusted for confounding variables. The series of model-based linkage analyses and TDTs provided inconclusive evidence on possible linkage of both phenotypes to the 11q 12-13 chromosomal region. Conclusions : We support the hypothesis of a major gene effect in heritability of hand osteoarthritis in both phenotypes. Despite the fact that some DNA markers showed statistically significant association to studied primary phenotypes, we find only weak evidence of linkage disequilibrium between hand osteoarthritis and the proximal part of the 11q 12-13 chromosomal segment (D11S1983 for K–L score and D11S1313 for OPS). The subject, however, a merit requires further investigation.

The distribution of plasma lipoprotein[a] (Lp[a] ) concentrations, a risk factor for cardiovascular disease, varies greatly among racial groups, with African Americans having values that are shifted toward higher levels than those of whites. The underlying cause of this heterogeneity is unknown, but a role for "trans-acting" factors has been hypothesized. This study used genetic linkage analysis to localize genetic factors influencing Lp[a] levels in African Americans that were absent in other populations; linkage results were analyzed separately in non-Hispanic whites, Hispanic whites, and African Americans. As expected, all three samples showed highly significant linkage at the approximate location of the lysophosphatidic acid locus. The white populations also independently had regions of significant linkage on chromosome 19 (LOD 3.80) and suggestive linkage on chromosomes 12 (LOD 1.60), 14 (LOD 2.56), and 19 (LOD 2.52).? No linkage evidence was found to support the hypothesis of another single gene with large effects specifically segregating in African Americans that may account for their elevated Lp[a] levels.-Barklej, R. A., A. C. Brown, C. L. Hanis, S. L. Kardia, S. T. Turner, and E. Boerwinkle. Lack of genetic linkage evidence for a trans-acting factor having a large effect on plasma lipoprotein[a] levels in African Americans.

Linkage of creatinine clearance to chromosome 10 in Utah pedigrees replicates a locus for end-stage renal disease in humans and renal failure in the fawn-hooded rat.BackgroundRenal failure is an important health concern for persons with hypertension and diabetes. In the fawn-hooded rat, a renal failure locus, Rf-1, has been identified on rat chromosome 1. A study of African American sibpairs with end-stage renal disease (ESRD) replicated this finding on the orthologous region in humans, chromosome 10, with a maximum logarithm of odds (LOD) score of 3.4. An important question is whether this region can be detected in healthy subjects prior to onset of ESRD by examining creatinine clearance as an indicator of early renal damage.MethodsWe analyzed 49 Utah Caucasian pedigrees and performed quantitative nonparametric linkage analysis using 21 markers spanning chromosome 10. Pedigree members (mean age of 40 ± 17) were examined up to three different times over 10 years, with creatinine clearance measured at each exam. For examination 1, three overnight, timed, 12-hour urine samples were obtained and averaged. One 12-hour sample was obtained for examinations 2 and 3.ResultsHeritabilities of creatinine clearance were 0.33 (N = 1360), 0.36 (N = 1196), and 0.53 (N = 718) for the three examinations, respectively. The nonparametric LOD score for examination 1 was 1.4 at marker D10S677 (∼117 cM). The LOD score at examination 2, an average of 21/2 years later, was 1.8 at marker D10S1239 (∼123 cM) and 1.9 at marker D10S1425 (∼137 cM). The LOD score at examination 3, an average of 10 years from baseline, was 2.1 at marker D10S2470 (∼113 cM). Thus, there is consistent evidence of linkage to this region from three different examinations spanning a period of 10 years.ConclusionsThese linkage results confirm the ESRD linkage and the rat renal failure linkage to this region even though the LOD score is somewhat weaker, probably due to the less severe phenotype that was analyzed. It also suggests that there may be a locus on chromosome 10 that leads to reduced renal function that can be detected while subjects are still healthy. Identification of the responsible gene may help in predicting renal disease progression in susceptible patients.

Further evidence for linkage of autosomal-dominant medullary cystic kidney disease on chromosome 1q21.BackgroundAutosomal-dominant medullary cystic kidney disease (ADMCKD) is characterized by the development of cysts at the corticomedullary border of the kidneys. It resembles nephronophthisis (NPH) with an autosomal-recessive mode of inheritance. Genetic linkage has been shown either on chromosome 1q21 (ADMCKD1) or 16p12 (ADMCKD2), and families exist who are not linked to the aforementioned loci. No disease-causing gene underlying this disorder has been reported.MethodsThe Finnish Transplantation Register and hospital records were searched to identify all of the ADMCKD families in the Finnish population. Detailed clinical information of the patients was collected. Linkage analysis was used to study whether the Finnish families originating from a homogenous population showed genetic linkage to the ADMCKD1 or ADMCKD2 loci. Also, the coding region of a strong candidate gene, natriuretic peptide receptor A (NPRA), located on the chromosome 1q21 critical region, was sequenced using polymerase chain reaction sequencing with an ABI 377XL Automated DNA sequencer (Applera Corp., Norwalk, CT, USA).ResultsFive of the six families showed linkage to the previously identified region of chromosome 1q21. Family 6 with hyperuricemia as a prominent clinical feature was linked to neither of the ADMCKD loci. Wide interfamiliar and intrafamiliar variability in the clinical picture of the patients was detected. The NPRA gene mutation was excluded as a causative gene by sequencing.ConclusionThis study locates the gene for ADMCKD1 close to a marker D1S1595 in a region

The Comel–Netherton syndrome is an autosomal recessive multisystemic disorder characterized by localized or generalized congenital ichthyosis, hair shaft abnormalities, immune deficiency, and markedly elevated IgE levels. Life-threatening complications during infancy include temperature and electrolyte imbalance, recurrent infections, and failure to thrive. To study the clinical presentations of the Comel–Netherton syndrome and its molecular cause, we ascertained 19 unrelated families of various ethnic backgrounds. Results of initial linkage studies mapped the Comel–Netherton syndrome in 12 multiplex families to a 12 cM interval on 5q32, thus confirming genetic homogeneity of Comel–Netherton syndrome across families of different origins. The Comel–Netherton syndrome region harbors the SPINK5 gene, which encodes a multidomain serine protease inhibitor (LEKTI) predominantly expressed in epithelial and lymphoid tissues. Recently, recessive mutations in SPINK5 were identified in several Comel–Netherton syndrome patients from consanguineous families. We used heteroduplex analysis followed by direct DNA sequencing to screen all 33 exons and flanking intronic sequences of SPINK5 in the affected individuals of our cohort. Mutation analysis revealed 17 distinct mutations, 15 of which were novel, segregating in 14 Comel–Netherton syndrome families. The nucleotide changes included four non-sense mutations, eight small deletions or insertions leading to frameshift, and five splice site defects, all of which are expected to result in premature terminated or altered translation of SPINK5. Almost half of the mutations clustered between exons 2 and 8, including two recurrent mutations. Genotype–phenotype correlations suggested that homozygous nucleotide changes resulting in early truncation of LEKT1 are associated with a severe phenotype. For the first time, we used molecular data to perform prenatal testing, thus demonstrating the feasibility of molecular diagnosis in the Comel–Netherton syndrome.

Medical geneticists connect relatives having the same disease into a family structure called a pedigree. Genetic linkage analysis uses pedigrees to find the approximate chromosomal locations of disease-causing genes. The problem of choosing a pedigree is particularly interesting for diseases inherited in an autosomal recessive pattern in inbred populations because there are many possible paths of inheritance to choose from. A variety of shortcuts are taken to produce plausible pedigrees from inbred populations. We lay the mathematical foundations for a shortcut that was recently used in a pedigree-disease study of an inbred Mennonite population. Recessive disease genes can be localized using the shortcut of homozygosity mapping by finding regions of the genome where affected persons are homozygous. An important quantity in homozygosity mapping is the inbreeding coefficient of a person, which is the prior probability that the person inherited the same piece of DNA on both copies of the chromosome from a single ancestor. Software packages are ill-suited to handle large pedigrees with many inbreeding loops. Therefore, we consider the problem of generating small pedigrees that match the inbreeding coefficient of one or more affected persons in the larger pedigree. We call such a problem an inverse inbreeding coefficient problem . We focus on the case where there is one sibship with one or more affected persons, and consider the problem of constructing a pedigree so that it is “simpler” and gives the sibship a specified inbreeding coefficient. First, we give a construction that yields small pedigrees for any inbreeding coefficient. Second, we add the constraint that ancestor-descendant matings are not allowed, and we give another more complicated construction to match any inbreeding coefficient. Third, we show some examples of how to use the one-sibship construction to do pedigree replacement on real pedigrees with multiple affected sibships. Fourth, we give a different construction to match the inbreeding coefficient of one sibship, while attempting to minimize a measure of the inbreeding loop complexity.

There have been recent reports of transmission-ratio distortion (TRD) or segregation distortion in families not selected for genetic disease. If TRD exists but is ignored, linkage studies searching for disease genes in affected relatives may be misinterpreted. We show that the identical-by-descent sharing patterns for affected sib pairs are strongly affected by TRD and, further, that the estimated statistical significance of a sib-pair linkage study may be extremely biased. However, we also show that, if TRD is suspected during the planning stage of a study, the planned sample size of the study needs to be increased by only a small amount to maintain the desired power.

Disseminated superficial actinic porokeratosis is an autosomal dominant cutaneous disorder characterized by many uniformly small, minimal, annular, anhidrotic, and keratotic lesions. The genetic basis for this disease is unknown. Using a genomewide search in a large Chinese family, we identified a locus at chromosome 12q23.2-24. 1 responsible for disseminated superficial actinic porokeratosis. The fine mapping study indicates that the disseminated superficial actinic porokeratosis gene is located within a 9.6 cM region between markers D12S1727 and D12S1605, with a maximum two-point LOD score of 20.53 (theta = 0.00) at D12S78. This is the first locus identified for a genetic disease where the major phenotype is porokeratosis. The study provides a map location for isolation of a gene causing disseminated superficial actinic porokeratosis.

XDH gene mutation is the underlying cause of classical xanthinuria: A second report. Background Classical xanthinuria is a rare autosomal recessive disorder characterized by excessive excretion of xanthine in urine. Type I disease results from the isolated deficiency of xanthine dehydrogenase (XDH), and type II results from dual deficiency of XDH and aldehyde oxidase. The XDH gene has been cloned and localized to chromosome 2p22-23. The aim of this study was to characterize the molecular basis of classical xanthinuria in an Iranian-Jewish family. Methods The apparently unrelated parents originated from a community in which consanguineous marriages are common. Subtyping xanthinuria was attempted by homozygosity mapping using microsatellite markers D2S352, D2S367, and D2S2374 in the vicinity of the XDH gene. Mutation detection was accomplished by PCR-SSCP screening of all 36 exons and exon-intron junctions of the XDH gene, followed by direct sequencing and confirmation of sequence alteration by restriction analysis. Results The index case was homozygous for all three microsatellite markers analyzed. The expected frequency of this genotype in a control population was 0.0002. These results suggested that xanthinuria in the patient is linked to the XDH gene. Consequently, a 1658insC mutation in exon 16 of the XDH gene was identified. The 1658insC mutation was not detected in 65 control DNA samples. Conclusion A molecular approach to the diagnosis of classical xanthinuria type I in a female patient with profound hypouricemia is described. Linkage of xanthinuria to the XDH locus was demonstrated by homozygosity mapping, and a 1658insC mutation, predicting a truncated inactive XDH protein, was identified. These results reinforce the notion that mutations in the XDH gene are the underlying cause of classical xanthinuria type I.

Extreme discordant sibling-pair (EDSP) designs have been shown in theory to be very powerful for mapping quantitative-trait loci (QTLs) in humans. However, their practical applicability has been somewhat limited by the need to phenotype very large populations to find enough pairs that are extremely discordant. In this paper, we demonstrate that there is also substantial power in pairs that are only moderately discordant, and that designs using moderately discordant pairs can yield a more practical balance between phenotyping and genotyping efforts. The power we demonstrate for moderately discordant pairs stems from a new statistical result. Statistical analysis in discordant-pair studies is generally done by testing for reduced identity by descent (IBD) sharing in the pairs. By contrast, the most commonly-used statistical methods for more standard QTL mapping are Haseman-Elston regression and variance-components analysis. Both of these use statistics that are functions of the trait values given IBD information for the pedigree. We show that IBD sharing statistics and “trait value given IBD” statistics contribute complementary rather than redundant information, and thus that statistics of the two types can be combined to form more powerful tests of linkage. We propose a simple composite statistic, and test it with simulation studies. The simulation results show that our composite statistic increases power only minimally for extremely discordant pairs. However, it boosts the power of moderately discordant pairs substantially and makes them a very practical alternative. Our composite statistic is straightforward to calculate with existing software; we give a practical example of its use by applying it to a Genetic Analysis Workshop (GAW) data set.

The identification of genes contributing to complex diseases and quantitative traits requires genetic data of high fidelity, because undetected errors and mutations can profoundly affect linkage information. The recent emphasis on the use of the sibling-pair design eliminates or decreases the likelihood of detection of genotyping errors and marker mutations through apparent Mendelian incompatibilities or close double recombinants. In this article, we describe a hidden Markov method for detecting genotyping errors and mutations in multilocus linkage data. Specifically, we calculate the posterior probability of genotyping error or mutation for each sibling-pair-marker combination, conditional on all marker data and an assumed genotype-error rate. The method is designed for use with sibling-pair data when parental genotypes are unavailable. Through Monte Carlo simulation, we explore the effects of map density, marker-allele frequencies, marker position, and genotype-error rate on the accuracy of our error-detection method. In addition, we examine the impact of genotyping errors and error detection and correction on multipoint linkage information. We illustrate that even moderate error rates can result in substantial loss of linkage information, given efforts to fine-map a putative disease locus. Although simulations suggest that our method detects ≤50% of genotyping errors, it generally flags those errors that have the largest impact on linkage results. For high-resolution genetic maps, removal of the errors identified by our method restores most or nearly all the lost linkage information and can be accomplished without generating false evidence for linkage by removing incorrectly identified errors.

There is a lot of confusion in the literature about the “differences” between “model-based” and “model-free” methods and about which approach is better suited for detection of the genes predisposing to complex multifactorial phenotypes. By starting from first principles, we demonstrate that the differences between the two approaches have more to do with study design than statistical analysis. When simple data structures are repeatedly ascertained, no assumptions about the genotype-phenotype relationship need to be made for the analysis to be powerful, since simple data structures admit only a small number of df. When more complicated and/or heterogeneous data structures are ascertained, however, the number of df in the underlying probability model is too large to have a powerful, truly “model-free” test. So-called “model-free” methods typically simplify the underlying probability model by implicitly assuming that, in some sense, all meioses connecting two affected individuals are informative for linkage with identical probability and that the affected individuals in a pedigree share as many disease-predisposing alleles as possible. By contrast, “model-based” methods add structure to the underlying parameter space by making assumptions about the genotype-phenotype relationship, making it possible to probabilistically assign disease-locus genotypes to all individuals in the data set on the basis of the observed phenotypes. In this study, we demonstrate the equivalence of these two approaches in a variety of situations and exploit this equivalence to develop more powerful and efficient likelihood-based analogues of “model-free” tests of linkage and/or linkage disequilibrium. Through the use of a “pseudomarker” locus to structure the space of observations, sib-pairs, triads, and singletons can be analyzed jointly, which will lead to tests that are more well-behaved, efficient, and powerful than traditional “model-free” tests such as the affected sib-pair, transmission/disequilibrium, haplotype relative risk, and case-control tests. Also described is an extension of this approach to large pedigrees, which, in practice, is equivalent to affected relative-pair analysis. The proposed methods are equally applicable to two-point and multipoint analysis (using complex-valued recombination fractions).

Although several genetic forms of rare or syndromic hypertriglyceridemia have been reported, little is known about the specific chromosomal regions across the genome harboring susceptibility genes for common forms of hypertriglyceridemia. Therefore, we conducted a genomewide scan for susceptibility genes influencing plasma triglyceride (TG) levels in a Mexican American population. We used both phenotypic and genotypic data from 418 individuals distributed across 27 low-income, extended Mexican American families. For the analyses, TG values were log transformed (ln TG). We used a variance-components technique to conduct multipoint linkage analyses for localizing susceptibility genes that determine variation in TG levels. We used an approximately 10-15-cM map, which was made on the basis of information from 295 microsatellite markers. After accounting for the effects of sex and sex-specific age terms, we found significant evidence for linkage (LOD = 3.88) of ln TG levels to a genetic location between the markers GABRB3 and D15S165 on chromosome 15q. This putative locus explains 39.7+/-7% (P=.000012) of total phenotypic variation in ln TG levels. Suggestive evidence was found for linkage of ln TG levels to two different locations on chromosome 7, which are approximately 85 cM apart from each other. Also, there is some evidence for linkage of high-density lipoprotein cholesterol concentrations to a genetic location near one of the regions on chromosome 7. In conclusion, we found strong evidence for linkage of ln TG levels to a genetic location on chromosome 15q in a Mexican American population, which is prone to disease conditions such as type 2 diabetes and the insulin-resistance syndrome that are associated with hypertriglyceridemia. This putative locus appears to have a major influence on ln TG variation.

Brachydactyly type A-1 (BDA1) was, in 1903, the first recorded example of a human anomaly with Mendelian autosomal dominant inheritance. Two large families, the affected members of which were radiographed, were recruited in the study we describe here. Two-point linkage analysis for pedigree 1 (maximum LOD score [Zmax] 6.59 at recombination fraction [theta] 0.00) and for pedigree 2 (Zmax=5.53 at straight theta=0.00) mapped the locus for BDA1 in the two families to chromosome 2q. Haplotype analysis of pedigree 1 confined the locus for family 1 within an interval of8.1 cM flanked by markers D2S2248 and D2S360, which was mapped to chromosome 2q35-q36 on the cytogenetic map. Haplotype analysis of pedigree 2 confined the locus for family 2 within an interval of28. 8 cM flanked by markers GATA30E06 and D2S427, which was localized to chromosome 2q35-q37. The two families had no identical haplotype within the defined region, which suggests that the two families were not related.

Familial nephrotic syndrome: Clinical spectrum and linkage to chromosome 19q13. Background Familial nephrotic syndrome (NS) has both autosomal dominant and recessive forms of inheritance. Recent studies in families with an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) have been at odds concerning linkage to chromosome 19q13 (Mathis et al, Kidney Int 53:282–286, 1998; Winn et al, Kidney Int 55:1241–1246, 1999), suggesting genetic heterogeneity. This study examines the clinical features and confirms linkage to chromosome 19q13 in a family with autosomal dominant NS. Methods DNA samples were obtained from 16 of 17 family members. Genomic DNA was isolated, and polymerase chain reaction was performed for five markers spanning the area of interest on chromosome 19q13. Data were evaluated using two- and six-point linkage analysis. Results Clinical features included presentation of NS in childhood, steroid unresponsiveness, and slow progression to renal failure. Renal biopsy in affected family members showed lesions ranging from minimal change to mesangial proliferative glomerulonephritis to FSGS. Linkage was confirmed between the disease state and chromosome 19q13, with a maximum logarithm of odds (LOD) score of 2.41. Linkage was observed for a 7 cM region on chromosome 19q13, defined by markers D19S425 and D19S220. Conclusions This study confirms the Mathis et al report of linkage to chromosome 19q13 in a family with autosomal dominant NS. However, there were notable differences in the presenting clinical and histopathologic features of our affected family members compared with those of Mathis et al. This suggests that the gene on chromosome 19q13 may be responsible for considerable phenotypic heterogeneity and variable expression in both clinical presentation and renal histopathology.

Summary Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance to a multitude of self-antigens. Epidemiological data suggest an important role for genes in the etiology of lupus, and previous genetic studies have implicated the HLA locus, complement genes, and low-affinity IgG (Fcγ) receptors in SLE pathogenesis. In an effort to identify new susceptibility loci for SLE, we recently reported the results of a genomewide microsatellite marker screen in 105 SLE sib-pair families. By using nonparametric methods, evidence for linkage was found in four intervals: 6p11-21 (near the HLA), 16q13, 14q21-23, and 20p12.3 (LOD scores ⩾2.0), and weaker evidence in another nine regions. We now report the results of a second complete genome screen in a new cohort of 82 SLE sib-pair families. In the cohort 2 screen, the four best intervals were 7p22 (LOD score 2.87), 7q21 (LOD score 2.40), 10p13 (LOD score 2.24), and 7q36 (LOD score 2.15). Eight additional intervals were identified with LOD scores in the range 1.00–1.67. A combined analysis of MN cohorts 1 and 2 (187 sib-pair families) showed that markers in 6p11-p21 (D6S426, LOD score 4.19) and 16q13 (D16S415, LOD score 3.85) met the criteria for significant linkage. Three intervals (2p15, 7q36, and 1q42) had LOD scores in the range 1.92–2.06, and another 13 intervals had LOD scores in the range of 1.00–1.78 in the combined sample. These data, together with other available gene mapping results in SLE, are beginning to allow a prioritization of genomic intervals for gene discovery efforts in human SLE.

SummaryHereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous disorders causing progressive spasticity and weakness of the lower limbs. We report a large family of French descent with autosomal dominant pure HSP. We excluded genetic linkage to the known loci causing HSP and performed a genomewide search. We found evidence for linkage of the disorder to polymorphic markers on chromosome 2q24-q34: a maximum LOD score of 3.03 was obtained for marker D2S2318. By comparison with families having linkage to the major locus of pure autosomal dominant HSP (SPG4 on chromosome 2p), there were significantly more patients without Babinski signs, with increased reflexes in the upper limbs, and with severe functional handicaps.

Recent studies suggest that hereditary prostate cancer (PRCA) is a complex disease, involving multiple susceptibility genes and variable phenotypic expression. Through linkage analysis, potential prostate cancer susceptibility loci have been mapped to 3 regions on chromosome 1. To investigate the reported linkage to these regions, we conducted linkage studies on 144 PRCA families by using microsatellite markers in regions 1q24-25 (HPC1) and 1q42.2-43 (PCAP). We also examined the 1p36 (CAPB) region in 13 PRCA families with at least one case of brain cancer. No significant evidence of linkage to the HPC1 or PCAP region was found when the entire data set was analyzed. However, weak evidence for linkage to HPC1 was observed in the subset of families with male-to-male transmission (n=102; maximum multipoint nonparametric linkage [NPL] 1.99, P=.03). Weak evidence for linkage with heterogeneity within this subset was also observed (HLOD 1.21, P=.02), with approximately 20% of families linked. Although not statistically significant, suggestive evidence for linkage to PCAP was observed for the families (n=21) that met the three criteria of male-to-male transmission, average age of diagnosis66 years, and/=5 affected individuals (maximum multipoint NPL 1.45, P=.08). There was no evidence for linkage to CAPB in the brain cancer-prostate cancer subset. These results strengthen the argument that prostate cancer is a heterogeneous disease and that multiple genetic and environmental factors may be important for its etiology.

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation. IBD is subdivided into Crohn disease and ulcerative colitis phenotypes. Given the immunologic dysregulation in IBD, the human-leukocyte-antigen region on chromosome 6p is of significant interest. Previous association and linkage analysis has provided conflicting evidence as to the existence of an IBD-susceptibility locus in this region. Here we report on a two-stage linkage and association analysis of both a basic population of 353 affected sibling pairs (ASPs) and an extension of this population to 428 white ASPs of northern European extraction. Twenty-eight microsatellite markers on chromosome 6 were genotyped. A peak multipoint LOD score of 4.2 was observed, at D6S461, for the IBD phenotype. A transmission/disequilibrium test (TDT) result of P=.006 was detected for D6S426 in the basic population and was confirmed in the extended cohort (P=.004; 97 vs. 56 transmissions). The subphenotypes of Crohn disease, ulcerative colitis, and mixed IBD contributed equally to this linkage, suggesting a general role for the chromosome 6 locus in IBD. Analysis of five single-nucleotide polymorphisms in the TNFA and LTA genes did not reveal evidence for association of these important candidate genes with IBD. In summary, we provide firm linkage evidence for an IBD-susceptibility locus on chromosome 6p and demonstrate that TNFA and LTA are unlikely to be susceptibility loci for IBD.

Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive disorder; major phenotypic findings include dysmorphic extremities, retinal dystrophy, obesity, male hypogenitalism, and renal anomalies. In the majority of northern European families with BBS, the syndrome is linked to a 26-cM region on chromosome 11q13. However, the finding, so far, of five distinct BBS loci (BBS1, 1q; BBS2, 16q; BBS3, 3p; BBS4, 15q; BBS5, 2q) has hampered the positional cloning of these genes. We use linkage disequilibrium (LD) mapping in an isolated founder population in Newfoundland to significantly reduce the BBS1 critical region. Extensive haplotyping in several unrelated BBS families of English descent revealed that the affected members were homozygous for overlapping portions of a rare, disease-associated ancestral haplotype on chromosome 11q13. The LD data suggest that the BBS1 gene lies in a 1-Mb, sequence-ready region on chromosome 11q13, which should enable its identification.

To the Editor: Diaphragmatic spinal muscular atrophy (SMA) has been delineated as a variant of infantile SMA (SMA1 [MIM 253300]) (Mellins et al. 1974; Bertini et al. 1989). The most prominent symptoms are severe respiratory distress resulting from diaphragmatic paralysis with eventration shown on chest x-ray and predominant involvement of the upper limbs and distal muscles. In contrast to classic SMA1, in diaphragmatic SMA the upper spinal cord is more severely affected than the lower section. The pmn mouse presents with progressive motor neuronopathy and a disease that closely resembles diaphragmatic SMA (Schmalbruch et al. 1991). The pmn locus has been mapped to murine chromosome 13 (Brunialti et al. 1995). Here we report on nine patients from three families with diaphragmatic SMA following autosomal recessive inheritance. The diagnosis of diaphragmatic SMA was made on the basis of clinical criteria (Rudnik-Schoneborn et al. 1996). Family 1 is of Lebanese origin; family 2, German origin; and family 3, Italian origin. We obtained DNA samples from these families after receiving informed consent, in accordance with the Declaration of Helsinki. In family 1 (fig. 1A), the parents are first cousins. The first affected son died, at the age of 10 wk, of suspected sudden infant death syndrome (SIDS). One daughter presented, at the age of 6 wk, with feeding difficulties and progressive respiratory distress. Chest x-ray showed eventration of the diaphragm. Mechanical ventilation was initiated at the age of 8 wk. She developed progressive muscular atrophy with complete paralysis of the upper and lower limbs and mild contractures of the knee and ankle joints. Three other children, nonidentical twin daughters and the youngest daughter, died of respiratory failure—the twins at the age of 8 and 9 wk and the youngest daughter at the age of 8 wk. Autopsy specimens were taken from gastrocnemius muscle in both twins and from the upper spinal cord in one twin. Skeletal-muscle histology revealed neurogenic atrophy without signs of reinnervation. Ultrastructurally, the motor end plates lacked nerve terminals and showed postsynaptic degenerative changes characterized by deep invaginations. The diameter of anterior spinal roots was reduced in the upper spinal cord. The remaining motor neurons showed chromatolysis. These findings offer two different pathophysiological concepts: (1) degeneration of the anterior horn cells of the spinal cord with neurogenic muscular atrophy suggests dying-forward atrophy, and (2) presynaptic and postsynaptic signs of motor end-plate degeneration suggest dying-back atrophy. In family 2 (fig. 1B), the first child had severe muscular hypotonia and died, at the age of 9 wk, of cardiorespiratory failure. The third child has been mechanically ventilated since the age of 3 mo. In family 3 (fig. 1C), which has been reported in detail elsewhere (Novelli et al. 1995), the gene locus for SMA1, on chromosome 5q, has been excluded. Both affected sibs presented with respiratory insufficiency right after birth and with the typical signs of diaphragmatic SMA. Figure 1 Haplotypes in families with diaphragmatic SMA subtypes. A, Family 1 (Lebanese origin): age at onset, 6–10 wk. B, Family 2 (German origin): age at onset, 9–12 wk. C, Family 3 (Italian origin): onset at birth. Haplotype analysis indicated ... First, we confirmed that, in families 1 and 2, there is no linkage of the trait to markers of the SMA locus on 5q11.2-q13.3, as there is in family 3. Second, the orthologous regions corresponding to the murine pmn gene region on human chromosomes 1q and 7p were excluded as gene loci responsible for the disease (Grohmann et al. 1998). To locate the gene locus for diaphragmatic SMA, a whole-genome scan was undertaken in family 1. Microsatellite analysis was performed, by standard semiautomated methods, by an ABI 377-Sequencer, and the results were processed by GENESCAN software, as described elsewhere (Saar et al. 1997). The whole-genome linkage scan was performed with the use of 340 polymorphic fluorescence–labeled markers spaced at ∼10-cM intervals throughout the autosomal part of the genome. Subsequent fine mapping was performed with eight additional microsatellite markers. Markers were chosen from the Genethon final linkage map. Two-point parametric linkage analyses were performed with the LINKAGE package, version 5.2 (Lathrop and Lalouel 1984), under the following assumptions: a regular, fully penetrant autosomal recessive trait locus with a disease-allele frequency of .002 and no phenocopy rate, codominant marker loci with uniformly distributed allele frequencies, and standard recombination rates. Multipoint analysis was performed with the GENEHUNTER program, version 1.3 (Kruglyak et al. 1996). Genomewide linkage scanning of family 1 revealed linkage of diaphragmatic SMA only to markers on chromosome 11q13-q21. In the following, we name this subtype of diaphragmatic SMA “spinal muscular atrophy with respiratory distress” (SMARD). For the markers D11S1296, D11S4095, D11S901, D11S1358, and D11S1757, a maximum two-point LOD score of 3.16 at recombination fraction (θ) 0 was obtained. The two-point LOD scores for 13 markers on chromosome 11q are summarized in table 1. Haplotype analysis revealed a recombination event in individual 2.4 that placed the disease locus distal to marker D11S1883 (fig. 1A). The crossing-over in individual 2.1 placed the disease locus proximal to marker D11S917. Consistent with parental consanguinity, all affected siblings from family 1 were autozygous for all markers within the cosegregating segment. Multipoint linkage analysis with the use of 13 markers yielded a maximum LOD score of 3.86, which clearly places the disease locus between D11S1883 and D11S917 (Genethon map positions 68.5 cM and 100.9 cM). Table 1 LOD-Score Values at Standard Recombination Rates for Markers on Chromosome 11q in Lebanese Family 1 In family 2, the two affected sibs shared two identical parental haplotypes in the SMARD cosegregating segment on 11q13-q21, a finding that supports the assignment of the SMARD locus to this region (fig. 1B). In family 3, haplotype analysis was inconsistent with linkage to the markers tested (fig. 1C). Thus, this locus was excluded as being responsible for the disease in this family. Our finding that diaphragmatic SMA with onset at age 6–12 wk is linked to chromosome 11q markers in two apparently unrelated families from different countries (families 1 and 2) but that diaphragmatic SMA with onset at birth does not show such linkage (family 3) suggests that diaphragmatic SMA is both clinically and genetically heterogeneous. The prevalence of diaphragmatic SMA is unknown. However, in a series of >200 patients with early-onset SMA, ∼1% presented with diaphragmatic SMA and did not have a deletion of the survival motor-neuron gene (SMN) on chromosome 5q (Rudnik-Schoneborn et al. 1996). Considering the case history of the affected son from family 1 who had suspected SIDS, we presume that some of those infants with SIDS may possibly have been misdiagnosed. We are currently looking for further patients with SMARD, to refine the large cosegregating region on chromosome 11q.

SummaryAlthough much theoretical work has been undertaken to derive thresholds for statistical significance in genetic linkage studies, real data are often complicated by many factors, such as missing individuals or uninformative markers, which make the validity of these theoretical results questionable. Many simulation-based methods have been proposed in the literature to determine empirically the statistical significance of the observed test statistics. However, these methods either are not generally applicable to complex pedigree structures or are too time-consuming. In this article, we propose a computationally efficient simulation procedure that is applicable to arbitrary pedigree structures. This procedure can be combined with statistical tests, to assess the statistical significance for genetic linkage between a locus and a qualitative or quantitative trait. Furthermore, the genomewide significance level can be appropriately controlled when many linked markers are studied in a genomewide scan. Simulated data and a diabetes data set are analyzed to demonstrate the usefulness of this novel simulation method.

Summary Schizophrenia is a severe mental disorder affecting ∼1% of the world's population. Here, we report the results from a three-stage genomewide screen performed in a study sample from an internal isolate of Finland. An effort was made to identify genes predisposing for schizophrenia that are potentially enriched in this isolate, which has an exceptionally high lifetime risk for this trait. Ancestors of the local families with schizophrenia were traced back to the foundation of the population in the 17th century. This genealogical information was used as the basis for the study strategy, which involved screening for alleles shared among affected individuals originating from common ancestors. We found four chromosomal regions with markers revealing pairwise LOD scores >1.0: 1q32.2-q41 ( Z max =3.82, dominant affecteds-only model), 4q31 ( Z max =2.74, dominant 90%-penetrance model), 9q21 ( Z max =1.95, dominant 90%-penetrance model), and Xp11.4-p11.3 ( Z max =2.01, recessive 90%-penetrance model). This finding suggests that there are several putative loci predisposing to schizophrenia, even in this isolate.

SummarySeveral methods have been proposed for linkage analysis of complex traits with unknown mode of inheritance. These methods include the LOD score maximized over disease models (MMLS) and the “nonparametric” linkage (NPL) statistic. In previous work, we evaluated the increase of type I error when maximizing over two or more genetic models, and we compared the power of MMLS to detect linkage, in a number of complex modes of inheritance, with analysis assuming the true model. In the present study, we compare MMLS and NPL directly. We simulated 100 data sets with 20 families each, using 26 generating models: (1) 4 intermediate models (penetrance of heterozygote between that of the two homozygotes); (2) 6 two-locus additive models; and (3) 16 two-locus heterogeneity models (admixture α = 1.0, .7, .5, and .3; α = 1.0 replicates simple Mendelian models). For LOD scores, we assumed dominant and recessive inheritance with 50% penetrance. We took the higher of the two maximum LOD scores and subtracted 0.3 to correct for multiple tests (MMLS-C). We compared expected maximum LOD scores and power, using MMLS-C and NPL as well as the true model. Since NPL uses only the affected family members, we also performed an affecteds-only analysis using MMLS-C. The MMLS-C was both uniformly more powerful than NPL for most cases we examined, except when linkage information was low, and close to the results for the true model under locus heterogeneity. We still found better power for the MMLS-C compared with NPL in affecteds-only analysis. The results show that use of two simple modes of inheritance at a fixed penetrance can have more power than NPL when the trait mode of inheritance is complex and when there is heterogeneity in the data set.

Summary We apply the method of “blocking Gibbs” sampling to a problem of great importance and complexity—linkage analysis. Blocking Gibbs sampling combines exact local computations with Gibbs sampling, in a way that complements the strengths of both. The method is able to handle problems with very high complexity, such as linkage analysis in large pedigrees with many loops, a task that no other known method is able to handle. New developments of the method are outlined, and it is applied to a highly complex linkage problem in a human pedigree.

SummaryMucolipidosis type IV (MLIV) is a lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. It is a rare autosomal recessive disease, and the majority of patients diagnosed, to date, are of Ashkenazi Jewish descent. We have mapped the MLIV gene to chromosome 19p13.2-13.3 by linkage analysis with 15 markers in 13 families. A maximum LOD score of 5.51 with no recombinants was observed with marker D19S873. Several markers in the linked interval also displayed significant linkage disequilibrium with the disorder. We constructed haplotypes in 26 Ashkenazi Jewish families and demonstrate the existence of two founder chromosomes in this population. The localization of MLIV to chromosome 19 will permit genetic prenatal diagnosis in affected families and will aid in the isolation of the disease gene.