MASSACHUSETTS GENERAL HOSPITAL, THE
Congenital Diaphragmatic Hernia (CDH) is a frequent and often fatal developmental condition of diaphragm defects associated with lung hypoplasia caused by diverse factors that we hypothesize are predominantly genetic, but heterogeneous. Project IV will use a combinationof clinical, molecular, developmental, genomic and cytogenetic strategies to identify mutations causing CDH, with the hope of elucidating perturbed biochemical pathways that can then serve as functional targets for pharmacological intervention or treatment. Aim I: We will recruit a cohort of carefully phenotyped isolated and complex CDH patients from two major cljnica! sites, MGHand Children's Hospital Boston, establish cell lines on each patient, and parents, and siblings, and enter deceased populations of patients, families with multiple affected members, consanguineous families, and monozygotic twins. DMA will be extracted from various sources for mutational analysis of candidate genes, arrayed based Comparative Genomic Hybridization (aCGH), loss of heterozygosity studies (LOH), and metaphase preparations for subtelomeric FISH. Aim II: High resolution cytogenetic tools such as 1 Mbarray CDH, subtelomeric FISH, and multiplex igand-directed probe amplification (MLPA) will be used to identify microdeletions, microduplications. and balanced and unbalanced translocations followed by breakpoint analyses to identify genes in these regions hat have mutations causative for CDH. Aim III: Nonsynonomous SNPs identified will be studied as plausible causative mutations. Genome wide SNP analysis will be used to reveal regions of LOH in consanguineous Donnai Barrow kindreds and in discordant monozygotic twins. Aim IV: Candidate genes from animal models or from LOH regions with CDH in human will be studied br abnormal expression in human CDH lungs or diaphragms and tested in Drosophila or mouse cell-based or organ culture assays to determine functional significance. CDH gene defects uncovered by these studies will serve as targets for pharmacological or other therapeutic interventionsto ameliorate or prevent this severe birth malformation.
| AWARD OVERVIEW |
| Award Number |
3R01HD055150-04S2 |
Funding Agency |
Department of Health and Human Services |
| Total Award Amount |
$124,146 |
Project Location - City |
Boston |
| Award Date |
09/22/2009 |
Project Location - State |
MA |
| Project Status |
Completed |
Project Location - Zip |
02114-0000
|
| Jobs Reported |
1.00 |
Congressional District |
09 |
| Project Location - Country |
US |
|
|
Recipient Information
(Grants)
| Recipient Information (Grants) |
|
Recipient Name
|
MASSACHUSETTS GENERAL HOSPITAL, THE |
| Recipient DUNS Number |
073130411
|
| Recipient Address |
55 FRUIT ST |
| Recipient City |
BOSTON |
| Recipient State |
Massachusetts |
| Recipient Zip |
02114-2621 |
| Recipient Congressional District |
09 |
| Recipient Country |
USA |
Required to Report Top 5
Highly Compensated Officials |
No |
Projects and Jobs Information
| Projects and Jobs Information |
| Project Title |
Gene Mutation and Rescue in Human Diaphragmatic Hernia |
| Project Status |
Completed |
| Final Project Report Submitted |
Yes |
| Project Activities Description |
General Medical and Surgical Hospitals |
| Quarterly Activities/Project Description |
Approved protocols and consent forms are current and allow individual exchanges between the Massachusetts General Hospital (Dr. Donahoe) and Dr. Al-Gazali (UAE) to identify families with multiple affected probands with either isolated congenital diaphragmatic hernia (CDH), Donnai-Barrow syndrome (DBS), or G.I. anomalies and their family members. We had initiated the IRB process together with Dr. Teebi to obtain an identical approved protocol in Qatar, but were unable to complete the process due to Dr. Teebi?s passing. Variants found in families recruited by Dr. Al-Gazali have been plated and are in process to be resequenced in the larger number of 400+ patients recruited for this study.
Under Dr. Donahoe?s tutelage, Faouzi Maalouf, MD, a Fellow in the Pediatric Surgical Research Laboratories, met in person with Drs. Bitar and Nemer at the American University of Beirut (AUB) in Lebanon and hand-delivered our CDH IRB protocol and consent forms to them to gain agreement from their groups to establish an approved protocol which also allows recruitment of patients with CDH and G.I. anomalies for our study. This will be in addition to the patients with heart anomalies who they already recruit at the medical center of American University. Dr. Faouzi Maalouf (from AUB, Lebanon) continues to work diligently in Dr. Donahoe?s laboratory to validate expressed genes involved in lung and diaphragm development, while training in clinical genetics of birth defects, although he remains unfunded. The ARRA funds have allowed us to seamlessly maintain our longstanding collaboration with our colleagues in the Middle East to enroll and to study patients and families who have CDH or G.I. anomalies.
Define genomic locations of human malformation loci.
Six high priority CDH candidate genes were sequenced on 5 patients from separate families recruited by Drs. AL-Gazali and Teebi. For these 5 affecteds, plus one additional affected, we also ran Affymetrix 6.0 SNP arrays. aCGH was |
| Jobs Created |
1.00 |
| Description of Jobs Created |
Project Description:
A fundamental hypothesis in human genetics is that common causes of genetic disorders are informed by rare monogenic mendelian families. This approach has also proven to be fruitful in our study of Congenital Diaphragmatic Hernia (CDH).
A unique family displaying autosomal dominant isolated CDH has been enrolled in the study by Dr. Barbara R. Pober, M.D. and Meaghan K. Russell, M.P.H, Ph.D.c. A combination of linkage analysis, high density array platforms, and exome sequencing have allowed us to make progress in the discovery of the causative gene in this multiplex family.
The kindred was studied by Affymetrix 6.0 SNP arrays using Merlin and Allegro linkage software packages, assuming autosomal dominant inheritance, 50% penetrance, and Caucasian ethnicity (S. Hill, Ph.D.). Although no single region provided conclusive evidence of linkage, as expected from the pedigree size and structure, 17 genomic regions had LOD scores = 1.0 (peak ~1.6) suggesting they were more likely to harbor the causative mutation.
We performed copy number analysis using both the new Agilent 1M arrays and the previously collected data from Affymetrix 6.0 (K. Darvishi, Ph.D. & C. Lee Ph.D.). Sequence variants and copy number information was then intersected with linkage regions (M. Longoni, M.D.) providing a total of 15 CNVs. Strikingly, a small deletion interval (~180kb) on chromosome 8q23 was shared among affecteds and obligate carriers. This loss was independently confirmed on Affymetrix 6.0 array data and by SYBR green qPCR. Furthermore, we did not find comparably-sized CNVs in the Database of Genomic Variants, in other normal CNV collections accessible online, or in 13000 individuals in a control cohort (M. Talkowsky, Ph.D. & M. Daly, Ph.D.).
The deletion maps within the the CDH-associated gene FOG2 (ZFPM2, OMIM #603693), likely introducing an in frame deletion of the putative repression domain. Independent evidence of FOG2 involvement in CDH comes from mouse models, as well as human studies. Expression studies at the mRNA (M. Longoni, M.D. & F. Maalouf, M.D.) and protein (M. Longoni, M.D. & M.K. Russell, M.P.H., Ph.D.c) level are currently underway in patient-specific lymphoblastoid cell lines.
We also performed whole-exome sequencing in two affected patients, The exome data is currently being analyzed to pinpoint the role of genetic modifiers on the observed incomplete penetrance.
Further progress has been made in analyzing microarray expression data of mouse embryonic developing diaphragm. Meaghan K. Russell M.P.H, Ph.D.c, in collaboration with Dr. Longoni, M.D., has previously generated the first genome-scale gene expression dataset of the wild-type C57BL/6J mouse developing diaphragm and has recently finished an articulated statistical analysis of a comparable size dataset obtained from Fog2-ko and Gata4-ki mice, both of which display CDH phenotypes. Since the two are part of a repression complex, we focused our attention on the variant genes shared or held in common common between the two mutant data sets and identified 8 candidate transcripts (most notably hemicentin, HMCN-1, an extracellular matrix protein) that significantly overlap between the Fog2-ko and the Gata4-ko datasets. The probability of observing this overlap in two independent random samples computed using a hypergeometric distribution is unlikely.
Expression array analyses of developing diaphragm tissue in mice has provided us with a way to dissect the molecular signature of the developing diaphragm mesoderm. We have applied an in vitro protocol and differentiated control and patient-specific iPS cells to mesodermal precursors, which in turn can be further differentiated to the myogenic, osteogenic, and chondrogenic lineages (Barberi et al. 2005; Barberi et al, 2007; Stavropoulos et al, 2009). Successful differentiation was determined by expression of CD73, a multilineage mesoderm commitment marker (Barberi et al. 2007; Evseenko et al. 2010), and al |
Purchaser Information
(Grants)
| Purchaser Information |
| Contracting Office ID |
Not Reported |
| Contracting Office Name |
Not Available |
| Contracting Office Region |
Not Available |
| TAS Major Program |
75-0840 |
| Award Information |
| Award Date |
09/22/2009 |
| Award Number |
3R01HD055150-04S2 |
| Order Number |
|
| Award Type |
Grants |
| Funding Agency ID |
75 |
| Funding Agency Name |
Department of Health and Human Services |
| Funding Office Name |
Not Available |
| Awarding Agency ID |
75 |
| Awarding Agency Name |
Department of Health and Human Services |
| Amount of Award |
$124,146 |
| Funds Invoiced/Received |
$124,146 |
| Expenditure Amount |
$124,146 |
| Infrastructure Expenditure Amount |
$0 |
| Infrastructure Purpose and Rationale |
Not Reported |
| Infrastructure Point of Contact Name |
Not Reported |
| Infrastructure Point of Contact Email |
Not Reported |
| Infrastructure Point of Contact Phone |
Not Reported |
| Infrastructure Point of Contact Address |
Not Reported |
| Infrastructure Point of Contact City |
Not Reported |
| Infrastructure Point of Contact State |
Not Reported |
| Infrastructure Point of Contact Zip |
Not Reported |
Product or Service Information
(Grants)
| Product or Service Information |
| Primary Activity Code |
622110 |
| Activity Description |
General Medical and Surgical Hospitals |
| Sub-Awards Information |
| Sub-awards to Organizations |
0 |
| Sub-award Amounts to Organizations |
$0 |
| Sub-Awards to Individuals |
0 |
| Sub-Award Amounts to Individuals |
$0 |
| Number of Sub-awards less than $25,000/award |
0 |
| Amount of Sub-awards less than $25,000/award |
$0 |
| Number of payments to vendors greater than $25,000 |
0 |
| Total Amount of payments to vendors greater than $25,000/award |
$0 |
| Number of payments to vendors less than $25,000/award |
32 |
| Total Amount of payments to vendors less than $25,000/award |
$7,436 |
| Location Information |
| Latitude, Longitude |
42º 21' 44",
-71º 4' 11" |
| Congressional District |
09 |
| Address 1 |
55 Fruit Street |
| Address 2 |
|
| City |
Boston |
| County |
Suffolk |
| State |
MA |
| Zip |
02114-0000 |
|
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