Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ~86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1(eyes2) mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1(eyes2) embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis.

Feathers have complex forms and are an excellent model to study the development and evolution of morphologies. Existing chicken feather mutants are especially useful for identifying genetic determinants of feather formation. This study focused on the gene F, underlying the frizzle feather trait that has a characteristic curled feather rachis and barbs in domestic chickens. Our developmental biology studies identified defects in feather medulla formation, and physical studies revealed that the frizzle feather curls in a stepwise manner. The frizzle gene is transmitted in an autosomal incomplete dominant mode. A whole-genome linkage scan of five pedigrees with 2678 SNPs revealed association of the frizzle locus with a keratin gene-enriched region within the linkage group E22C19W28_E50C23. Sequence analyses of the keratin gene cluster identified a 69 bp in-frame deletion in a conserved region of KRT75, an α-keratin gene. Retroviral-mediated expression of the mutated F cDNA in the wild-type rectrix qualitatively changed the bending of the rachis with some features of frizzle feathers including irregular kinks, severe bending near their distal ends, and substantially higher variations among samples in comparison to normal feathers. These results confirmed KRT75 as the F gene. This study demonstrates the potential of our approach for identifying genetic determinants of feather forms.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

X-linked congenital hypertrichosis syndrome is associated with interchromosomal insertions mediated by a human-specific palindrome near SOX3.

X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.

Impact of restricted marital practices on genetic variation in an endogamous Gujarati group.

Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries-old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with groups outside the gol. Gujarati Patels practice this form of "exogamic endogamy." We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non-Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y-chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low-level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine-scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation.

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.

Prevalence of common disease-associated variants in Asian Indians.

BACKGROUND: Asian Indians display a high prevalence of diseases linked to changes in diet and environment that have arisen as their lifestyle has become more westernized. Using 1200 genome-wide polymorphisms in 432 individuals from 15 Indian language groups, we have recently shown that: (i) Indians constitute a distinct population-genetic cluster, and (ii) despite the geographic and linguistic diversity of the groups they exhibit a relatively low level of genetic heterogeneity. RESULTS: We investigated the prevalence of common polymorphisms that have been associated with diseases, such as atherosclerosis (ALOX5), hypertension (CYP3A5, AGT, GNB3), diabetes (CAPN10, TCF7L2, PTPN22), prostate cancer (DG8S737, rs1447295), Hirschsprung disease (RET), and age-related macular degeneration (CFH, LOC387715). In addition, we examined polymorphisms associated with skin pigmentation (SLC24A5) and with the ability to taste phenylthiocarbamide (TAS2R38). All polymorphisms were studied in a cohort of 576 India-born Asian Indians sampled in the United States. This sample consisted of individuals whose mother tongue is one of 14 of the 22 "official" languages recognized in India as well as individuals whose mother tongue is Parsi, a cultural group that has resided in India for over 1000 years. Analysis of the data revealed that allele frequency differences between the different Indian language groups were small, and interestingly the variant alleles of ALOX5 g.8322G>A and g.50778G>A, and PTPN22 g.36677C>T were present only in a subset of the Indian language groups. Furthermore, a latitudinal cline was identified both for the allele frequencies of the SNPs associated with hypertension (CYP3A5, AGT, GNB3), as well as for those associated with the ability to taste phenylthiocarbamide (TAS2R38). CONCLUSION: Although caution is warranted due to the fact that this US-sampled Indian cohort may not represent a random sample from India, our results will hopefully assist in the design of future studies that investigate the genetic causes of these diseases in India. Our results also support the inclusion of the Indian population in disease-related genetic studies, as it exhibits unique genotype as well as phenotype characteristics that may yield new insights into the underlying causes of common diseases that are not available in other populations.

Inherited dental anomalies: a review and prospects for the future role of clinicians.

Inherited dental anomalies such as hypodontia, supernumerary teeth, enamel defects, and diastema are evident in large segments of most populations. Although treatment options for many of these conditions are ever improving, much remains to be understood about their etiology and pathophysiology. In this review, the authors hope to enthuse dental professionals into aiding the human geneticist by collaborating in studies seeking the underlying genetic cause of dental anomalies and referring patients presenting these conditions to the human geneticist.

Gene discovery for dental anomalies: a primer for the dental professional.

BACKGROUND: Thousands of inherited human disorders have been catalogued to date, but the underlying genetic causes of less than 20 percent of those disorders have been discovered. TYPE OF STUDIES REVIEWED: The completion of the Human Genome Project (HGP) has made available the DNA sequence of all 24 human chromosomes, thereby allowing the localization of all human genes and, ultimately, determination of their function. Disease gene discovery is being expedited greatly by the data from the HGP, thereby paving the way for determination of the genetic etiology of most of these disorders. RESULTS: While most dental anomalies can severely affect patients' quality of life, they are not fatal, which makes multigenerational families with these disorders available for study. These families are invaluable for genetic studies. Despite this fact, the discovery of genes underlying non-syndromic dental anomalies has lagged behind that for anomalies affecting other organ systems. The authors present an overview of the methodologies of disease gene identification using hypodontia, which is one of the most common anomalies of the dentition, to illustrate the application of these principles. CLINICAL IMPLICATIONS: An understanding of the advances in human genetics should inspire the practicing dental professional to ascertain whether a dental anomaly is inherited and, if so, work with a human geneticist to identify its underlying genetic mechanism.

A simple method for DNA isolation from clotted blood extricated rapidly from serum separator tubes.

BACKGROUND: After clinical laboratory tests have been performed, it can be difficult to obtain DNA without further patient involvement. Although the blood clot remaining within the serum-separation tube after serum collection is a source of DNA, recovery of the clot from the tube is a significant challenge. METHOD: We devised a method to efficiently remove clotted blood from the serum-separation gel and extract DNA from clotted whole blood samples, obtaining maximum yield of the DNA without DNA contamination by the separation gel. The method involved centrifugation of the sample in the inverted original 10-mL collection tube to displace the separation gel for easy isolation of the blood clot and shearing of the blood clot by centrifugation through a 20-gauge wire mesh cone at 2000 g in a swinging-bucket rotor. After erythrocyte lysis and proteinase-K digestion of the fragmented clot, DNA was precipitated with isopropanol in the presence of glycogen. RESULTS: The mean amount of DNA obtained from a 4-mL clotted blood sample prepared by this method was 37.1 microg for clots processed soon after collection, with a reduction to 0.439 microg for clots stored for 1 month before extraction. The quality of the DNA was comparable to that extracted directly from whole blood, and it was found to be suitable for PCR-mediated analysis. CONCLUSION: We have formulated a method that overcomes the difficulties of safely extricating a blood clot from serum-separation tubes, allowing rapid DNA extraction for the purposes of genetic investigation.

Identification of novel genes expressed during mouse tooth development by microarray gene expression analysis.

To identify genes heretofore undiscovered as critical players in the biogenesis of teeth, we have used microarray gene expression analysis of the developing mouse molar tooth (DMT) between postnatal day (P) 1 and P10 to identify genes differentially expressed when compared with 16 control tissues. Of the top 100 genes exhibiting increased expression in the DMT, 29 were found to have been previously associated with tooth development. Differential expression of the remaining 71 genes not previously associated with tooth development was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. Further analysis of seven of the latter genes by mRNA in situ hybridization found that five were specific to the developing tooth in the craniofacial region (Rspo4, Papln, Amtn, Gja1, Maf). Of the remaining two, one was found to be more widely expressed (Sp7) and the other was found to be specific to the nasal serous gland, which is close to, but distinct from, the developing tooth (Vrm).

A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3.

Amelogenesis imperfecta (AI) is a collective term used to describe phenotypically diverse forms of defective tooth enamel development. AI has been reported to exhibit a variety of inheritance patterns, and several loci have been identified that are associated with AI. We have performed a genome-wide scan in a large Brazilian family segregating an autosomal dominant form of AI and mapped a novel locus to 8q24.3. A maximum multipoint LOD score of 7.5 was obtained at marker D8S2334 (146,101,309 bp). The disease locus lies in a 1.9 cM (2.1 Mb) region according to the Rutgers Combined Linkage-Physical map, between a VNTR marker (at 143,988,705 bp) and the telomere (146,274,826 bp). Ten candidate genes were identified based on gene ontology and microarray-facilitated gene selection using the expression of murine orthologues in dental tissue, and examined for the presence of a mutation. However, no causative mutation was identified.

Nonconventional genetic risk factors for cardiovascular disease.

Despite numerous advances made in identifying the genes for rare mendelian forms of cardiovascular disease (CVD), relatively little is known about the common, complex forms at the genetic level. Moreover, most genes that have been associated with CVD, whether they are single gene forms or more common forms of the disease, have primarily been involved in biochemical pathways related to what are considered "conventional" risk factors. However, recent genetic studies have begun to identify genes and pathways associated with CVD that would not be considered to underlie conventional risk factors. In this review, we discuss the evidence for this latter notion based on recent linkage and association studies in humans. As an example, we also illustrate how a combination of mouse and human genetics led to identification of the 5-lipoxygenase pathway for CVD, with potentially important implications for its treatment and diagnosis. We conclude with a discussion of the prospects for identifying CVD genes in the future and for potentially developing more effective therapeutic strategies.

Genes underlying familial hypodontia: a review and discussion of the role of dental hygienists in future research.

Congenitally missing teeth, or hypodontia, is one of the most common abnormalities of the human dentition and has a critical and often lifelong impact on the oral health of affected individuals. Here we review hypodontia and describe the patterns of inheritance it can display. A short review of tooth development and a primer in human genetics are presented. Approaches used to determine the underlying cause for various forms of hypodontia are discussed and information about genes discovered to date is reviewed. The role that the dental hygienists can play in facilitating the discovery of novel genes for hypodontia is illustrated.

Examination of ancestry and ethnic affiliation using highly informative diallelic DNA markers: application to diverse and admixed populations and implications for clinical epidemiology and forensic medicine.

We and others have identified several hundred ancestry informative markers (AIMs) with large allele frequency differences between different major ancestral groups. For this study, a panel of 199 widely distributed AIMs was used to examine a diverse set of 796 DNA samples including self-identified European Americans, West Africans, East Asians, Amerindians, African Americans, Mexicans, Mexican Americans, Puerto Ricans and South Asians. Analysis using a Bayesian clustering algorithm (STRUCTURE) showed grouping of individuals with similar ethnic identity without any identifier other than the AIMs genotyping and showed admixture proportions that clearly distinguished different individuals of mixed ancestry. Additional analyses showed that, for the majority of samples, the predicted ethnic identity corresponded with the self-identified ethnicity at high probability (P > 0.99). Overall, the study demonstrates that AIMs can provide a useful adjunct to forensic medicine, pharmacogenomics and disease studies in which major ancestry or ethnic affiliation might be linked to specific outcomes.

Comparative analysis of Schwann cell lines as model systems for myelin gene transcription studies.

Primary and immortalized cultured Schwann cells are commonly utilized in analyses of myelin gene promoters, but few lines are well-characterized in terms of their endogenous expression of myelin genes. This is particularly significant in that cultured Schwann cells typically do not express myelin genes at levels comparable to those observed in vivo. In this study, the steady-state levels of mRNA and protein for five Schwann cell markers (PMP22, P0, MBP, MAG, and LNGF-R) were assessed in primary Schwann cells and six representative Schwann cell lines (RT4-D6P2T, JS-1, RSC96, R3, S16, and S16Y). RT4-D6P2T and S16 cells were the most similar to myelinating Schwann cells based on their comparatively high expression of PMP22 and P0 mRNA. Both RT4-D6P2T and S16 also expressed P0 protein. In addition, the previously reported P1-A positive regulatory region from the myelination-specific PMP22 promoter demonstrated significant activity in both these cell lines. However, nuclear proteins that interacted with P1-A were different in extracts prepared from RT4-D6P2T and S16 cells. Primary Schwann cells expressed myelin proteins at levels that were equal or less than those observed with the RT4-D6P2T and S16 lines, indicating that primary Schwann cells are not necessarily better than immortalized Schwann cells as model systems for the study of myelin gene regulation. The data presented here demonstrate that cultured Schwann cells used to study myelin gene promoters have to be carefully selected on the basis of the endogenous level of expression of the myelin gene under study.

Novel missense mutations and a 288-bp exonic insertion in PAX9 in families with autosomal dominant hypodontia.

We describe the molecular analysis of three families with hypodontia involving primarily molar teeth and report two novel mutational mechanisms. Linkage analysis of two large families revealed that the hypodontia was linked to the PAX9 locus. These two families revealed missense mutations consisting of a glutamic acid substitution for lysine and a proline substitution for leucine within the paired domain of PAX9. A pair of identical twins affected with hypodontia in a third family demonstrated a 288-bp insertion within exon 2 that resulted in a putative frameshift mutation and a premature stop codon. The insertion was associated with the loss of 7-bp from exon 2. A block of 256-bp of sequence within the insertion was completely identical to downstream sequence from the second intron of the PAX9 gene. These studies extend the spectrum of mutations in PAX9 associated with hypodontia to include heretofore undescribed categories, including missense mutations.

Reciprocal crossovers and a positional preference for strand exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2.

Smith-Magenis syndrome (SMS) is caused by an approximately 4-Mb heterozygous interstitial deletion on chromosome 17p11.2 in approximately 80%-90% of affected patients. Three large ( approximately 200 kb), complex, and highly homologous ( approximately 98%) low-copy repeats (LCRs) are located inside or flanking the SMS common deletion. These repeats, also known as "SMS-REPs," are termed "distal," "middle," and "proximal." The directly oriented distal and proximal copies act as substrates for nonallelic homologous recombination resulting in both the deletion associated with SMS and the reciprocal duplication: dup(17)(p11.2p11.2). Using restriction enzyme cis-morphism analyses and direct sequencing, we mapped the regions of strand exchange in 16 somatic-cell hybrids that harbor only the recombinant SMS-REP. Our studies showed that the sites of crossovers were distributed throughout the region of homology between the distal and proximal SMS-REPs. However, despite approximately 170 kb of high homology, 50% of the recombinant junctions occurred in a 12.0-kb region within the KER gene clusters. DNA sequencing of this hotspot (positional preference for strand exchange) in seven recombinant SMS-REPs narrowed the crossovers to an approximately 8-kb interval. Four of them occurred in a 1,655-bp region rich in polymorphic nucleotides that could potentially reflect frequent gene conversion. For further evaluation of the strand exchange frequency in patients with SMS, novel junction fragments from the recombinant SMS-REPs were identified. As predicted by the reciprocal-recombination model, junction fragments were also identified from this hotspot region in patients with dup(17)(p11.2p11.2), documenting reciprocity of the positional preference for strand exchange. Several potential cis-acting recombination-promoting sequences were identified within the hotspot. It is interesting that we found 2.1-kb AT-rich inverted repeats flanking the proximal and middle KER gene clusters but not the distal one. The role of any or all of these in stimulating double-strand breaks around this positional recombination hotspot remains to be explored.