Clinical sequencing of the retinitis pigmentosa gene RPGR in over 1,000 cases of vision loss.

RPGR pathogenic variants are the major cause of X-linked retinitis pigmentosa. Here, we report the results from 1,033 clinical DNA tests that included sequencing of RPGR. A total of 184 RPGR variants were identified: 78 pathogenic or likely pathogenic, 14 uncertain, and 92 likely benign or benign. Among the pathogenic and likely pathogenic variants, 23 were novel, and most were frameshift or nonsense mutations (87%) and enriched (67%) in RPGR exon 15 (ORF15). Identical pathogenic variants found in different families were largely on different haplotype backgrounds, indicating relatively frequent, recurrent RPGR mutations. None of the 16 mother/affected son pairs showed de novo mutations; all 16 mothers were heterozygous for the pathogenic variant. These last two observations support the occurrence of most RPGR mutations in the male germline.

Structural basis for the disruption of the cerebral cavernous malformations 2 (CCM2) interaction with Krev interaction trapped 1 (KRIT1) by disease-associated mutations.

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1(NPX(Y/F)3), revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1(NPX(Y/F)3). We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.

Mutant deoxynucleotide carrier is associated with congenital microcephaly.

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.

Linkage disequilibrium between STRPs and SNPs across the human genome.

Patterns of linkage disequilibrium (LD) reveal the action of evolutionary processes and provide crucial information for association mapping of disease genes. Although recent studies have described the landscape of LD among single nucleotide polymorphisms (SNPs) from across the human genome, associations involving other classes of molecular variation remain poorly understood. In addition to recombination and population history, mutation rate and process are expected to shape LD. To test this idea, we measured associations between short-tandem-repeat polymorphisms (STRPs), which can mutate rapidly and recurrently, and SNPs in 721 regions across the human genome. We directly compared STRP-SNP LD with SNP-SNP LD from the same genomic regions in the human HapMap populations. The intensity of STRP-SNP LD, measured by the average of D', was reduced, consistent with the action of recurrent mutation. Nevertheless, a higher fraction of STRP-SNP pairs than SNP-SNP pairs showed significant LD, on both short (up to 50 kb) and long (cM) scales. These results reveal the substantial effects of mutational processes on LD at STRPs and provide important measures of the potential of STRPs for association mapping of disease genes.

A founder mutation in the Ashkenazi Jewish population affecting messenger RNA splicing of the CCM2 gene causes cerebral cavernous malformations.

PURPOSE: Cerebral cavernous malformations can occur sporadically or are caused by mutations in one of three identified genes. Cerebral cavernous malformations often remain clinically silent until a mutation carrier suffers a stroke or seizure. Presymptomatic genetic testing has been valuable to follow and manage cerebral cavernous malformation mutation carriers. During routine diagnostic testing, we identified a two base pair change in seven unrelated people of Ashkenazi Jewish heritage. Because of the location of the variant beyond the invariant splice donor sequence, the change was reported as a variant of unknown significance. In this study, we determined whether this change was a disease-causing mutation and whether it represents a founder mutation in the Ashkenazi Jewish population. METHODS: Transcripts arising from the normal and mutant alleles were examined by reverse transcription-polymerase chain reaction from affected and unaffected Ashkenazi Jewish cerebral cavernous malformation family members. A synthetic splicing system using a chimeric exon was used to visualize the effects of the change on splice donor site utilization. RESULTS: The two base pair change in CCM2, c.30 + 5_6delinsTT, segregated with affected status in the study families. Reverse transcription-polymerase chain reaction revealed loss of the transcript allele that was in phase with the mutation. The two base pair change, when tested in an in vitro synthetic splicing system, altered splice donor site utilization. Resequencing of the genomic region proximal and distal to the CCM2 gene mutation revealed a common single-nucleotide polymorphism haplotype in affected individuals. CONCLUSIONS: The two base pair change in CCM2, c.30 + 5_6delinsTT, disrupted proper splice donor utilization leading to a degraded transcript. Single nucleotide polymorphism haplotype analysis demonstrated that this mutation was due to a founder in the Ashkenazi Jewish population. These data have the potential to simplify genetic testing for cerebral cavernous malformation in the Ashkenazi Jewish population.

The genetic structure of Pacific Islanders.

Human genetic diversity in the Pacific has not been adequately sampled, particularly in Melanesia. As a result, population relationships there have been open to debate. A genome scan of autosomal markers (687 microsatellites and 203 insertions/deletions) on 952 individuals from 41 Pacific populations now provides the basis for understanding the remarkable nature of Melanesian variation, and for a more accurate comparison of these Pacific populations with previously studied groups from other regions. It also shows how textured human population variation can be in particular circumstances. Genetic diversity within individual Pacific populations is shown to be very low, while differentiation among Melanesian groups is high. Melanesian differentiation varies not only between islands, but also by island size and topographical complexity. The greatest distinctions are among the isolated groups in large island interiors, which are also the most internally homogeneous. The pattern loosely tracks language distinctions. Papuan-speaking groups are the most differentiated, and Austronesian or Oceanic-speaking groups, which tend to live along the coastlines, are more intermixed. A small "Austronesian" genetic signature (always <20%) was detected in less than half the Melanesian groups that speak Austronesian languages, and is entirely lacking in Papuan-speaking groups. Although the Polynesians are also distinctive, they tend to cluster with Micronesians, Taiwan Aborigines, and East Asians, and not Melanesians. These findings contribute to a resolution to the debates over Polynesian origins and their past interactions with Melanesians. With regard to genetics, the earlier studies had heavily relied on the evidence from single locus mitochondrial DNA or Y chromosome variation. Neither of these provided an unequivocal signal of phylogenetic relations or population intermixture proportions in the Pacific. Our analysis indicates the ancestors of Polynesians moved through Melanesia relatively rapidly and only intermixed to a very modest degree with the indigenous populations there.

Low levels of genetic divergence across geographically and linguistically diverse populations from India.

Ongoing modernization in India has elevated the prevalence of many complex genetic diseases associated with a western lifestyle and diet to near-epidemic proportions. However, although India comprises more than one sixth of the world's human population, it has largely been omitted from genomic surveys that provide the backdrop for association studies of genetic disease. Here, by genotyping India-born individuals sampled in the United States, we carry out an extensive study of Indian genetic variation. We analyze 1,200 genome-wide polymorphisms in 432 individuals from 15 Indian populations. We find that populations from India, and populations from South Asia more generally, constitute one of the major human subgroups with increased similarity of genetic ancestry. However, only a relatively small amount of genetic differentiation exists among the Indian populations. Although caution is warranted due to the fact that United States-sampled Indian populations do not represent a random sample from India, these results suggest that the frequencies of many genetic variants are distinctive in India compared to other parts of the world and that the effects of population heterogeneity on the production of false positives in association studies may be smaller in Indians (and particularly in Indian-Americans) than might be expected for such a geographically and linguistically diverse subset of the human population.

Quantifying the increase in average human heterozygosity due to urbanisation.

The human population is undergoing a major transition from a historical metapopulation structure of relatively isolated small communities to an outbred structure. This process is predicted to increase average individual genome-wide heterozygosity (h) and could have effects on health. We attempted to quantify this increase in mean h. We initially sampled 1001 examinees from a metapopulation of nine isolated villages on five Dalmatian islands (Croatia). Village populations had high levels of genetic differentiation, endogamy and consanguinity. We then selected 166 individuals with highly specific personal genetic histories to form six subsamples, which could be ranked a priori by their predicted level of outbreeding. The measure h was then estimated in the 166 examinees by genotyping 1184 STR/indel markers and using two different computation methods. Compared to the value of mean h in the least outbred sample, values of h in the remaining samples increased successively with predicted outbreeding by 0.023, 0.038, 0.058, 0.067 and 0.079 (P<0.0001), where these values are measured on the same scale as the inbreeding coefficient (but opposite sign). We have shown that urbanisation was associated with an average increase in h of up to 0.08-0.10 in this Croatian metapopulation, regardless of the method used. Similar levels of differentiation have been described in many populations. Therefore, changes in the level of heterozygosity across the genome of this magnitude may be common during isolate break-up in humans and could have significant health effects through the established genetic mechanism of hybrid vigour/heterosis.

Channel glass-based detection of human short insertion/deletion polymorphisms by tandem hybridization.

The development and critical evaluation of new technologies for identifying genetic polymorphisms will rapidly accelerate the discovery and diagnosis of disease-related genes. We report a novel way for distinguishing a new class of human DNA polymorphisms, short insertion/deletion polymorphisms (indels). A sensor with cylindrical pores named channel glass in combination with tandem hybridization, which uses a 5'-fluorescent labeled stacking probe and microarray-based short allele-specific oligonucleotide (capture probe) was investigated. This methodology allows indels to be detected individually and rapidly with small quantities of target DNA. This establishes a reliable quantitative test. Approaches for simultaneously hybridizing different targets to arrayed probes, designed to detect various indels in parallel, were examined. Five markers were consistently detected in a single hybridization. Possible factors impeding the hybridization reaction process are discussed.

A comparison of hybridization efficiency between flat glass and channel glass solid supports.

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene.

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.

Paternal uniparental isodisomy of the entire chromosome 3 revealed in a person with no apparent phenotypic disorders.

Uniparental disomy (UPD) is a rare genetic abnormality. During a whole genome linkage study we identified a case of paternal uniparental isodisomy 3 serendipitously. This is the first ascertained human paternal UPD for chromosome 3 (UPD3pat). The finding of this paternal UPD case of the entire chromosome 3 with no apparent phenotypic disorders suggests that there are no paternal imprinted genes causing rare genetic disorders on chromosome 3.

STRP screening sets for the human genome at 5 cM density.

BACKGROUND: Short tandem repeat polymorphisms (STRPs) are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium) often require higher STRP density. RESULTS: We report the development of two separate 10 cM human STRP Screening Sets (Sets 12 and 52) which span all chromosomes. When combined, the two Sets contain a total of 782 STRPs, with average STRP spacing of 4.8 cM, average heterozygosity of 0.72, and total sex-average coverage of 3535 cM. The current Sets are comprised almost entirely of STRPs based on tri- and tetranucleotide repeats. We also report correction of primer sequences for many STRPs used in previous Screening Sets. Detailed information for the new Screening Sets is available from our web site: http://research.marshfieldclinic.org/genetics. CONCLUSION: Our new human STRP Screening Sets will improve the quality and cost effectiveness of genotyping for gene mapping and other applications.

A novel locus for adolescent idiopathic scoliosis on chromosome 12p.

Adolescent idiopathic scoliosis (AIS) is a common disorder with strong evidence for genetic predisposition. Quantitative trait loci (QTLs) for AIS susceptibility have been identified on chromosomes. We performed a genome-wide genetic linkage scan in seven multiplex families using 400 marker loci with a mean spacing of 8.6 cM. We used Genehunter Plus to generate linkage statistics, expressed as homogeneity (HLOD) scores, under dominant and recessive genetic models. We found a significant linkage signal on chromosome 12p, whose support interval extends from near 12 pter, spanning approximately 10 million bases or 31 cM. Fine mapping within the region using 20 additional markers reveals maximum HLOD = 3.7 at 5 cM under a dominant inheritance model, and a split peak maximum HLOD = 3.2 at 8 and 18 cM under a recessive inheritance model. The linkage support interval contains 95 known genes. We found evidence suggestive of linkage on chromosomes 1, 6, 7, 8, and 14. This study is the first to find evidence of an AIS susceptibility locus on chromosome 12. Detection of AIS susceptibility QTLs on multiple chromosomes in this and other studies demonstrate that the condition is genetically heterogeneous.

Genome-wide linkage screen for stature and body mass index in 3.032 families: evidence for sex- and population-specific genetic effects.

Stature (adult body height) and body mass index (BMI) have a strong genetic component explaining observed variation in human populations; however, identifying those genetic components has been extremely challenging. It seems obvious that sample size is a critical determinant for successful identification of quantitative trait loci (QTL) that underlie the genetic architecture of these polygenic traits. The inherent shared environment and known genetic relationships in family studies provide clear advantages for gene mapping over studies utilizing unrelated individuals. To these ends, we combined the genotype and phenotype data from four previously performed family-based genome-wide screens resulting in a sample of 9.371 individuals from 3.032 African-American and European-American families and performed variance-components linkage analyses for stature and BMI. To our knowledge, this study represents the single largest family-based genome-wide linkage scan published for stature and BMI to date. This large study sample allowed us to pursue population- and sex-specific analyses as well. For stature, we found evidence for linkage in previously reported loci on 11q23, 12q12, 15q25 and 18q23, as well as 15q26 and 19q13, which have not been linked to stature previously. For BMI, we found evidence for two loci: one on 7q35 and another on 11q22, both of which have been previously linked to BMI in multiple populations. Our results show both the benefit of (1) combining data to maximize the sample size and (2) minimizing heterogeneity by analyzing subgroups where within-group variation can be reduced and suggest that the latter may be a more successful approach in genetic mapping.

The genetic structure and history of Africans and African Americans.

Africa is the source of all modern humans, but characterization of genetic variation and of relationships among populations across the continent has been enigmatic. We studied 121 African populations, four African American populations, and 60 non-African populations for patterns of variation at 1327 nuclear microsatellite and insertion/deletion markers. We identified 14 ancestral population clusters in Africa that correlate with self-described ethnicity and shared cultural and/or linguistic properties. We observed high levels of mixed ancestry in most populations, reflecting historical migration events across the continent. Our data also provide evidence for shared ancestry among geographically diverse hunter-gatherer populations (Khoesan speakers and Pygmies). The ancestry of African Americans is predominantly from Niger-Kordofanian (approximately 71%), European (approximately 13%), and other African (approximately 8%) populations, although admixture levels varied considerably among individuals. This study helps tease apart the complex evolutionary history of Africans and African Americans, aiding both anthropological and genetic epidemiologic studies.

Effects of genome-wide heterozygosity on a range of biomedically relevant human quantitative traits.

The dramatic changes in human population structure over the last 200 years have resulted in significant levels of outbreeding, which, in turn, is predicted to lead to increased levels of individual genetic diversity (genome-wide heterozygosity, h). To investigate possible effects of these large demographic changes on global health, we studied the effect of h, measured as relative heterozygosity, h(R), on 15 disease-related traits in four groups of individuals with widely differing ancestral histories (ranging from outbred to inbred) from the Dalmatian islands in Croatia. Higher levels of h(R), estimated using 1184 STR/indel markers, were found in the outbred group (P < 0.0001) and were associated with lower blood pressure (BP) and total/LDL cholesterol (P = 0.01 and 0.01, respectively) after controlling for other factors, with BP showing a strong sex effect (males P > 0.5 and females P = 0.002). These findings, if replicated, suggest that h(R) be considered as a genetic risk factor in genetic epidemiological studies on common disease traits. They are consistent with the well-known effects of heterosis (hybrid vigour) described when outcrossing animals and plants. Outbreeding resulting from urbanization and migration from traditional population subgroups may be leading to increasing h(R) and may have beneficial effects on a range of traits associated with human health and disease. Other traits, such as age at menarche, IQ and lifespan, which have been changing during the decades of urbanization, may also have been influenced by demographic factors.

Clinical applications of Genome Polymorphism Scans.

Applications of Genome Polymorphism Scans range from the relatively simple such as gender determination and confirmation of biological relationships, to the relatively complex such as determination of autozygosity and propagation of genetic information throughout pedigrees. Unlike nearly all other clinical DNA tests, the Scan is a universal test--it covers all people and all genes. In balance, I argue that the Genome Polymorphism Scan is the most powerful, affordable clinical DNA test available today.