Philip F. Giampietro

Mutations in Transforming Growth Factor-β Receptor Type II Cause Familial Thoracic Aortic Aneurysms and Dissections

Background—A genetic predisposition for progressive enlargement of thoracic aortic aneurysms leading to type A dissection (TAAD) is inherited in an autosomal-dominant manner in up to 19% of patients, and a number of chromosomal loci have been identified for the condition. Having mapped a TAAD locus to 3p24–25, we sequenced the gene for transforming growth factor-&bgr; receptor type II (TGFBR2) to determine whether mutations in this gene resulted in familial TAAD. Methods and Results—We sequenced all 8 coding exons of TGFBR2 by using genomic DNA from 80 unrelated familial TAAD cases. We found TGFBR2 mutations in 4 unrelated families with familial TAAD who did not have Marfan syndrome. Affected family members also had descending aortic disease and aneurysms of other arteries. Strikingly, all 4 mutations affected an arginine residue at position 460 in the intracellular domain, suggesting a mutation “hot spot” for familial TAAD. Despite identical mutations in the families, assessment of linked polymorphisms suggested that these families were not distantly related. Structural analysis of the TGFBR2 serine/threonine kinase domain revealed that R460 is strategically located within a highly conserved region of this domain and that the amino acid substitutions resulting from these mutations will interfere with the receptor’s ability to transduce signals. Conclusion—Germline TGFBR2 mutations are responsible for the inherited predisposition to familial TAAD in 5% of these cases. Our results have broad implications for understanding the role of TGF-&bgr; signaling in the pathophysiology of TAAD.

Marshfield Clinic Personalized Medicine Research Project (PMRP): design, methods and recruitment for a large population-based biobank

OBJECTIVESnThe objective of this paper is to summarize the planning for Phase I of the Marshfield Clinic Personalized Medicine Research Project (PMRP) and to describe the recruitment efforts in the first 2 years.nnnMETHODSnThe purpose of Phase I of the PMRP was to develop a large population-based biobank with DNA, plasma and serum samples to facilitate genomics research. Planning and consultation was facilitated with three external boards: the Ethics and Security Advisory Board; the Scientific Advisory Board; and the Community Advisory Group. Commencing in September 2002, residents aged 18 and above who resided in 1 of 19 zip codes surrounding Marshfield, WI, USA, were invited to participate. After providing written informed consent, participants completed brief questionnaires that included questions about demographics, some environmental exposures, family history of disease, and adverse drug reactions, as well as family members living in the study area. Participants provided 50xa0ml of blood from which DNA was extracted and plasma and serum samples were stored. The informed consent document allowed access to electronic medical records and included language about non-disclosure of personal research results. A tick-off box was also included so that participants could either allow or decline subsequent recontact for future research studies.nnnRESULTSnA total of 17,463 subjects were enrolled during the first 23xa0months of recruitment (44.3% of the residents who the Research Project Assistants were able to contact). The participants ranged in age from 18 to 98.5xa0years (mean = 48.9, median = 48); 57.2% (n = 9986) were female. Self-reported race in the study cohort was similar to the year 2000 census for Wood County, WI, USA, with the majority (98%) reporting themselves to be White Caucasian. The majority of subjects (nxa0= 13,391, 76.7%) indicated that they had German ancestry. Only 142xa0participants (< 1%) opted out on the consent form for contact for future studies. The majority of the cohort reported that their current area of residence was a suburb, city or village (n = 10630, 60.87%); the remainder reported residence in a rural home or hobby farm (nxa0=xa05365, 30.72%), or a working farm or ranch (n = 1451, 8.31%). More than half the cohort (n = 9409, 53.88%) had lived on a working farm at some point in their life.nnnCONCLUSIONnThe PMRP database will allow research in three areas: genetic epidemiology, pharmacogenetics, and population genetics. The size and the stability of the population as well as the relative ethnic homogeneity will help facilitate longitudinal studies with valid research results that are not biased by population stratification.

Re: Human Papillomavirus DNA and p53 Polymorphisms in Squamous Cell Carcinomas From Fanconi Anemia Patients

Fanconi anemia is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, and the development of squamous cell carcinomas (SCCs) and other cancers. Recent clinicopathologic evidence has raised the possibility that an environmental factor such as human papillomavirus (HPV) may be involved in the pathogenesis of SCCs in Fanconi anemia patients. Given the high prevalence of p53 mutations in SCCs among the general population and the lack of p53 mutations in HPV-related carcinogenesis, we evaluated the role of HPV and p53 mutations and polymorphisms in SCC from Fanconi anemia patients. We used polymerase chain reaction (PCR) screening and real-time PCR to detect and quantify HPV DNA in DNA extracted from microdissected SCCs obtained from 24 Fanconi anemia patients (n = 25 SCCs; case subjects) and 50 age-, sex-, and tumor site-matched SCC patients without Fanconi anemia (n = 50 SCCs; control subjects). We PCR-amplified and sequenced exons 4-9 of the p53 gene from SCC DNA. We detected HPV DNA in 84% of the SCC specimens from the case subjects and in 36% of the SCC specimens from the control subjects (P<.001). The prevalence of p53 mutations in SCCs from the case subjects (0%, 0/25) was statistically significantly lower than that of SCCs from the control subjects (36%, 12/33; P<.001). A greater proportion of patients with Fanconi anemia and SCC were homozygous for Arg72, a p53 polymorphism that may be associated with increased risk for HPV-associated human malignancies, than an ethnically-matched cohort of Fanconi anemia patients without SCC (75% versus 51%; P =.05). These data suggest that Fanconi anemia is associated with increased susceptibility to HPV-induced carcinogenesis.

Abnormal vertebral segmentation and the notch signaling pathway in man.

Abnormal vertebral segmentation (AVS) in man is a relatively common congenital malformation but cannot be subjected to the scientific analysis that is applied in animal models. Nevertheless, some spectacular advances in the cell biology and molecular genetics of somitogenesis in animal models have proved to be directly relevant to human disease. Some advances in our understanding have come through DNA linkage analysis in families demonstrating a clustering of AVS cases, as well as adopting a candidate gene approach. Only rarely do AVS phenotypes follow clear Mendelian inheritance, but three genes—DLL3, MESP2, and LNFG—have now been identified for spondylocostal dysostosis (SCD). SCD is characterized by extensive hemivertebrae, trunkal shortening, and abnormally aligned ribs with points of fusion. In familial cases clearly following a Mendelian pattern, autosomal recessive inheritance is more common than autosomal dominant and the genes identified are functional within the Notch signaling pathway. Other genes within the pathway cause diverse phenotypes such as Alagille syndrome (AGS) and CADASIL, conditions that may have their origin in defective vasculogenesis. Here, we deal mainly with SCD and AGS, and present a new classification system for AVS phenotypes, for which, hitherto, the terminology has been inconsistent and confusing. Developmental Dynamics 236:1456–1474, 2007.

A novel microdeletion at 16p11.2 harbors candidate genes for aortic valve development, seizure disorder, and mild mental retardation.

Many multiple congenital anomalies (MCA) are caused by recombination between homologous segmental duplications. In this report, we describe a novel “de novo” microdeletion in male monozygotic twins presenting with aortic valve abnormality, seizure disorder, and mild mental retardation. Using array based comparative genomic hybridization, we mapped the microdeletion to the short arm of chromosome 16 at 16p11.2 and refined it using hemizygosity mapping to about 593 kb, a region that overlaps with 24 genes. The most probable mechanism for this microdeletion is through a specific intrachromosomal recombination between two, nearly identical, segmental duplications each spanning 147 kb that are flanking the microdeletion. Based on the phenotypes presented in the twins and what is known about the genes within the 16p11.2 microdeletion, we identified several genes that are strong candidates for the normal development of the aortic valve, as well as the development of seizure disorder and mental retardation.

Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal phenotypes

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.

Informed consent and subject motivation to participate in a large, population-based genomics study: the Marshfield Clinic Personalized Medicine Research Project.

Background: The objective of this study was to measure subject perspective and reaction to participation in the Personalized Medicine Research Project (PMRP) and to identify factors predicting understanding of the study elements. Method: Self-administered questionnaires were mailed to 1,593 subjects (10% sample). The questionnaire had three sections: section A consisted of 21 factual questions; section B consisted of 14 questions to assess the level of understanding about the PMRP concepts, and section C asked about the purpose of the PMRP. Results: The mean age of the 924 survey respondents was 52 years (SD = 16.9), with a range of 18–95 years. The majority of participants were female (n = 561, 61%). The percent of total correct responses for section A was significantly higher for females compared with males (males: 58.4% and females: 60.4%, t test = –2.18, p = 0.03) and age was significantly inversely related to percent of correct responses (β coefficient = –0.122, p < 0.001). More than one third of the participants indicated that the USD 20 greatly influenced their decision to participate in the project. In a multiple logistic regression model, people living outside of Marshfield were significantly more likely to indicate that the USD 20 greatly influenced their decision to participate (odds ratio = 1.40, 95% confidence limit = 1.06, 1.86) and age was inversely related to the monetary influence on decision to participate (odds ratio = 0.98, 95% confidence limit = 0.97, 0.98). Conclusion: Future community consultation efforts should highlight areas of lower understanding. In addition, research coordinators may need to take more time informing males and older individuals about project details so that they are making truly informed decisions about study participation.

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 12pter, spanning approximately 10 million bases or 31 cM. Fine mapping within the region using 20 additional markers reveals maximum HLODu2009=u20093.7 at 5 cM under a dominant inheritance model, and a split peak maximum HLODu2009=u20093.2 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.

Subtelomeric deletions of chromosome 9q: A novel microdeletion syndrome

Fluorescent in situ hybridization (FISH) screening of subtelomeric rearrangements has resulted in the identification of previously unrecognized chromosomal causes of mental retardation with and without dysmorphic features. This article reports the phenotypic and molecular breakpoint characterization in a cohort of 12 patients with subtelomeric deletions of chromosome 9q34. The phenotypic findings are consistent amongst these individuals and consist of mental retardation, distinct facial features and congenital heart defects (primarily conotruncal defects). Detailed breakpoint mapping by FISH, microsatellite and single nucleotide polymorphism (SNP) genotyping analysis has narrowed the commonly deleted region to an approximately 1.2 Mb interval containing 14 known transcripts. The majority of the proximal deletion breakpoints fall within a 400 kb interval between SNP markers C12020842 proximally and C80658 distally suggesting a common breakpoint in this interval.

Clinical genetics provider real-time workflow study

Purpose: Our work is the first documentation, in real time, of workflow in a general genetics department including data on patient care, research, and other activities for both clinical geneticists and genetic counselors.Methods: All physician geneticists and genetic counselors in the medical genetics department used an electronic tool to record their activities in 15 minute increments during clinic hours, evenings, and weekends over a 10-week period.Results: The average work week was 54.1 hours for physicians and 43.5 hours for genetic counselors. During clinic hours physicians spent about one-fourth of their time on direct patient care, one-fourth on other patient-related activities, one-fourth on research unrelated to individual patient care, and the remaining fourth on all other activities. However, after hours and on weekends they spent most of their time on research. Genetic counselors spent half of their time on patient-related activities, one-fourth on direct patient care, and the remainder on all other activities. The total professional time averaged 7 hours per new patient and 3.5 hours per follow-up with nearly 60% of this time devoted to patient-related activities.Conclusions: The labor intensive nature of clinical genetics, the large amount of time devoted to patient-related activities, and continuing limitations on billing by genetic counselors all contribute to the financial challenges faced by genetics departments.