clinical genomics
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Owen M. Rennert, MD, Head, Section on
Developmental Genomics Wai-yee
Chan, PhD, Adjunct Investigator Shao-Ming
Wu, PhD, Staff Scientist Margarita
Raygada, PhD, Staff Genetic Counselor Cigdem
F. Dogulu, MD, PhD, Clinical Fellow Sergei
Kvasha, PhD, Postdoctoral Fellow Michael
Y.K. Leung, PhD, Postdoctoral Fellow Alan
L.Y. Pang, PhD, Postdoctoral Fellow Queen
P. Vong, PhD, Postdoctoral Fellow Vanessa
Baxendale, MS, Research Associate Jeffrey
Benson, MS, Research Associate Diana
Alba, BS, Postbaccalaureate Fellow Deborah
Bellan, BS, Postbaccalaureate Fellow Warren
Johnson, BS, Postbaccalaureate Fellow Lisa
Ruszczyk, BS, Postbaccalaureate Fellow Evelyn
Law, BS, Summer Medical Student Angele
Nalbandian, BS, Graduate Student Deondra Simons, BS, Howard Hughes Student |
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Given that it involves mitosis, meiosis, and post-meiotic
differentiation, spermatogenesis provides an ideal system for studying the
intricate regulatory mechanism of cellular proliferation and differentiation.
We have obtained the profile of the genes expressed in mitotic, meiotic, and
post-meiotic germ cells and thus can take advantage of a rich resource for
studying development-specific mechanisms of gene expression. To understand
the change in gene expression from primordial germ cells to differentiated
spermatids, we mapped the transcriptome of embryonic gonads at different
stages of development; that work will provide information about genetic
regulation during early gonad development. An alternative approach is to
study the consequences of perturbation of normal development, such as in the
case of genetic mutations. Thus, we investigated the effects of a mutated
luteinizing hormone receptor in cultured cells as well as in intact animals.
In addition, our clinical protocols study pediatric patients with genetic and
metabolic disorders, giving us access to various genetic disorders as well as
providing our fellows with clinical genetics training. One such clinical
protocol concerns the identification of the role of susceptibility to
thrombosis in the pseudotumor cerebri of nephropathic cystinosis, which has
led to the development of a screen for mutational combinations in several
venous thrombosis-related molecules. Studies
of differentially expressed genes in spermatogenesis Pang, Johnson, Rennert, Chan; in
collaboration with Dym Using Serial Analysis of Gene Expression
(SAGE), we profiled the transcriptome of male germ cells at different stages
of development, i.e., mitosis, meiosis, and post-meiosis, as represented by
type A spermatogonia, pachytene spermatocytes, and round spermatids,
respectively. We deposited the SAGE data in our publicly accessible Website
(http://www.nichddirsage.nichd.nih.gov/publicsage/). Using 15,000-cDNA microarrays from the
National Institute on Aging, we also profiled expressed genes in pachytene
spermatocyte and round spermatid. Based on the similarity in changes in
profile of germ cells demonstrated by both techniques, we selected a number
of genes for further study, hypothesizing that the distinct expression
pattern of a gene reflects its specific role in different stages of
spermatogenesis. An X-linked gene, Testis
expressed gene 13 (Tex13), was
expressed predominantly in type A spermatogonia. Further studies showed that
a potential antisense transcript of Tex13,
complementary to the 3´ end of the sense transcript, is present and that the
expression pattern of the sense and antisense transcripts is similar but that
the relative expression level of the transcripts differs at different stages
of germ cells, suggesting that the expression of these transcripts is potentially
subject to post-transcriptional regulation by a mechanism such as gene
silencing. The regulation of expression, the biological activities, and the
relationship between the sense and antisense transcripts of Tex13 are currently under study. In a similar study, we found that, similar to Tex13, the mouse Lin-28 homolog is preferentially expressed during the mitotic
stage. In contrast, SAGE analysis of the embryonic male gonad indicated that
the expression patterns of the two transcripts differ during early gonadal
development. Previous investigations in worms demonstrated that Lin-28 is responsible for
developmental timing regulation and that its expression is subject to the
action of microRNA. We speculate that Tex13
and Lin-28 exert their effects
early in spermatogenesis. To elucidate the functional roles of the genes and
the mechanisms of gene expression regulation, we plan to generate gene
knockout mouse models. Based on microarray analysis, we have cloned a
novel isoform of the mouse Ard-1
gene, which encodes a putative co-subunit of murine N-terminal
acetyltransferase 1 complex. We found this novel Ard-1 transcript expressed predominantly in meiotic male germ
cells but not in nontesticular tissues, suggesting that the encoded product
may exert a meiotic stage–specific effect. From the mouse genome
sequence database, we identified the X-linked Ard-1 gene and another homologous transcript present on the
autosomes. Surprisingly, the latter two transcripts are universally expressed
but, during spermatogenesis, display expression patterns that differ from the
novel Ard-1 transcript. The
presence of a long unique 3´ untranslated region may subject the novel Ard-1 transcript to a germ
cell–specific mode of regulation of expression. The open reading frames
of the three translated products, although highly homologous, differ slightly
from each other. Further characterization of the novel Ard-1 protein is under
way to elucidate its role during spermatogenesis. Pang ALY, Taylor HC, Johnson W, Alexander S,
Chen Y, Su YA, Li X, Ravindranath N, Dym M, Rennert OM, Chan WY.
Identification of differentially expressed genes in mouse spermatogenesis. J Androl 2003;24:59-71. Antisense
transcription in differentiating germ cells Wu, Ruszczyk, Law, Baxendale, Pang, Rennert,
Chan; in collaboration with Stitely Antisense transcripts have been shown to be
involved in transcriptional and post-transcriptional gene regulation,
including genomic imprinting, X-inactivation, RNAi, RNA editing, and mRNA
processing, splicing, stability, transport, and translation. A recent
computational analysis identified 2,500 pairs of putative sense-antisense
transcripts out of 60,770 full-length mouse cDNA (about 8 percent). An
earlier study reported the identification of about 1,600 sense-antisense
transcriptional units (SATs) in the human genome. With the exception of the
antisense transcript of the SPEER2
gene, no other example of sense-antisense transcript pairs in mammalian germ
cell has been reported. Given that SAGE libraries are derived from
transcripts, the SAGE database of mouse type A spermatogonia, pachytene
spermatocytes, and round spermatids, as previously generated by our
laboratory, offers the opportunity to examine the presence of antisense
transcripts in these cells. Examination of 62 differentially expressed genes
identified in the three types of germ cell showed the presence of antisense
transcripts of 41 genes (66 percent). Various types of antisense transcript
can be identified, as shown in Figure 9.1. Of these 41 genes, 35 have
overlapping SATs. Nine of the genes have more than one SAT pair; 12 have
nonoverlapping antisense bidirectional transcripts (NABTs); and seven have
both SATs and NABTs. Using orientation-specific RT-PCR, we confirmed 29 genes
with an appreciable number of SAGE tags among the 41 genes. We confirmed the
presence of antisense transcripts experimentally for 17 genes. We compared
relative levels of expression of the SATs by quantitative real-time RT-PCR.
We examined tissue distribution of the SATs of the nine genes Uba52, Calm2, Ppp1cc, Ppic, Tsg1, Tcte3, Pdcl2, Prm 1, and Prm2 and
observed a wide spectrum of tissue-specific expression of SATs. The antisense
transcripts of four genes, namely Uba52, Tcte3, Prm1, and Prm2, were
cloned and characterized. Alignment of the nucleotide sequence of the
antisense transcripts with the genomic sequence of the genes encoding the
sense transcripts allowed localization of the antisense transcripts to exons
and introns of the sense gene and to pseudogene, intronic, and intergenic
sequences. A number of antisense transcripts contain appreciable open reading
frames that could be encoding novel proteins. The data show that antisense
transcription occurs more frequently in differentiating germ cells than in
somatic cells. We are identifying in
vitro and in vivo systems
suitable for testing functional activities of the cloned antisense
transcripts.
Wu SM, Baxendale V, Chen
Y, Li X, Pang ALY, Stitely T, Munson PJ, Leung MYK, Ravindranath N, Dym M,
Rennert OM, Chan WY. Analysis of mouse germ cell
transcriptome at different stages of spermatogenesis: biological
significance. Genomics
2004;84:971-981. Novel
activity of cytochrome c oxidase in germ cell development Wu, Ruszczyk, Johnson, Baxendale, Law,
Rennert, Chan Extensive apoptosis occurs during
spermatogenesis, particularly in spermatogonia and spermatocytes, but the
mechanism has not been fully defined. Recent studies suggested that
cytochrome c plays a critical role in germ cell apoptosis. Cytochrome c
exists in loosely and tightly bound pools attached to the inner
mitochrondrial membrane by association with cardiolipin, a complex that must
first be disrupted to generate a soluble pool of this protein. Once
cytochrome c is soluble, permeabilization of the outer mitochondrial membrane
with calcium or Bax is sufficient to allow extrusion of the protein into the
cytosol, resulting in the onset of apoptosis. Analysis of our germ cell SAGE
database showed the presence of the testis-specific cytochrome c with the
same differential expression pattern as the testis-specific isoform
cytochrome c oxidase VIb-2. Cloning of the transcript corresponding to the
most abundant novel tag in the three SAGE libraries showed that the
transcript corresponds to mouse cytochrome c oxidase subunit-3 (Cox 3). We
cloned a further abundant novel tag, which corresponded to another subunit of
cytochrome c oxidase (subunit 1, Cox 1). A search of the germ cell SAGE
libraries revealed that all 13 subunits of cytochrome c oxidase, complex IV
of the respiratory chain, are expressed at appreciable levels and demonstrate
comparable differential expression patterns in these cells. On the other
hand, some of the subunits of complexes II, III, and V of the respiratory
chain are absent, and only 12 of the 43 subunits of complex I have the same
differential expression pattern. The results imply that cytochrome c oxidase
may not function as a component of the respiratory chain in germ cells. We
hypothesize that cytochrome c oxidase oxidizes cytochrome c, causing its
release from the inner mitochrondrial membrane. Transfer of the soluble
cytochrome c into the cytosol results in amplification of calcium-dependent
apoptosis. We are in the process of testing this hypothesis by using in vitro cell models. Functional
genomic studies of gonad development and sexual dimorphism of the brain Baxendale, Vong, Bellan, Alba, Rennert,
Chan; in collaboration with Lau, Su To understand the mechanisms that regulate the
transition of primordial germ cells to gonocytes and the initiation of sexual
dimorphism, we are profiling the genes expressed in embryonic gonads of the
mouse. We are using SAGE to examine male and female embryonic gonads at
embryonic day 10.5 (E10.5), E11.5, E12.5, E13.5, E15.5, and E17.5 and the
mesonephros at E13.5, E15.5, and E17.5. We have completed an analysis of
about 152,000 SAGE tags for each of the male E10.5, E11.5, and E12.5 gonads.
The tags identify 24,460, 214,762, and 26,378 genes in the E10.5, E11.5, and
E12.5 gonads, respectively. The 10 most abundant tags represent cytochrome
b-245 beta polypeptide (Cybb), Cyp2e1 cytochrome P450 (COX5b),
Translationally controlled tumor protein (Tctp1), hemoglobin Y beta-like
embryonic chain (Hbb-y), tubulin alpha 2 (Tuba2), four ribosomal proteins
(X-linked S4, L26, 29, and L10A), and one uncharacterized cDNA. The gene
encoding embryonic hemoglobins beta and that encoding the X-linked ribosomal
protein S4 are either absent from or expressed at a very low level in germ
cells, indicating that both are specific for activities of the embryonic
gonads. Among the 10 most abundant tags present in embryonic gonads but
absent from germ cells, four represent genes encoding hemoglobin chains,
namely, Hba-X, Hbb-b1, Hbb-Y, and Hba-a1. The role of the hemoglobin genes in
early embryonic gonad development is not presently understood. However, a
recent paper that examined gene expression in embryonic lens also
demonstrated the expression of hemoglobin isoforms (Hba-a1, Hba-X, Hbb-b1,
Hbb-b2, and Hbb-Y). Even though fewer tags were sequenced for the germ cell
SAGE libraries (about 111,000 tags for each germ cell type versus about
152,000 tags for each embryonic gonad stage), we found (Wu SM et al., 2004) more specific genes
in germ cells than in embryonic gonads (4,946 germ cell specific-gene tags
versus 4,755 embryonic gonad specific-gene tags). These preliminary
observations have important implications for the regulation of gonad
development and germ cell differentiation. We are continuing our analysis of
expressed genes in male gonads at later embryonic ages and in female gonads
and mesonephros. We are interested in exploring the role of sex
chromosome–linked genes in sexual dimorphism of the brain. We have
generated human cDNA sex-linked gene microarrays of 724 X-linked and 28
Y-linked human genes on glass slides. We will use the microarrays to profile
expressed genes in the brain and gonad of male and female mice at E10.5,
E13.5, E15.5, E17.5, newborn mice, and adult mice. We will compare the
expression profile of the male and the female brain and gonad at different
time points and then examine the relationship between gonad development and
onset of sexual dimorphism of the brain. Wu SM, Baxendale V, Chen
Y, Li X, Pang ALY, Stitely T, Munson PJ, Leung MYK, Ravindranath N, Dym M,
Rennert OM, Chan WY. Analysis of mouse germ cell
transcriptome at different stages of spermatogenesis: biological
significance. Genomics
2004;84:971-981. The
role of Ddx3y in mouse gonad and
germ cell development Vong, Wu, Rennert, Chan; in collaboration
with Lau, Dym Among Y-encoded genes, Ddx3y (formerly known as Dby) has been considered a strong
candidate as a mediator of Y chromosome function in spermatogonial
proliferation. The tag representing Ddx3y
is present in the spermatogonial SAGE library. We subsequently cloned
full-length Ddx3y cDNA from mouse
type A spermatogonia in the form of two transcripts that differ only in the
length of the 3´untranslated region due to the presence of a different
polyadenylation signal. Both the long form (Dby-L) and the short form (Dby-S)
of Ddx3y are ubiquitously expressed
in nontesticular tissues except the ovary, with Dby-L expressed especially in brain and heart. We observed a
higher level of expression of Dby-L
and Dby-S in type A spermatogonia
than in spermatocytes and spermatids. In addition to the two variants, we
found two other types of alternatively spliced transcripts containing
nonoverlapping sequences of 48 and 120 nucleotides, respectively. We compared the expression of Ddx3y and its X and autosomal homologs
Ddx3 and D1Pas1 (formerly known as PL10)
in gonads at E10.5 with 18, 24, and 30 days after birth. Expression of Ddx3y rises from E10.5 to E17.5 and then
declines slowly after birth. In embryonic gonads, expression of Ddx3 is five- to 10-fold higher than
that of Ddx3y but drops after
birth. It has been reported that D1Pas1
is expressed only in pachytene spermatocytes and round spermatids, and we
noted its first expression at postnatal day 18, reaching its highest level
from day 24 to 30, when spermatocytes and spermatids first appear. Alignment
of Ddx3y, Ddx3, and D1Pas1 reveals
significant homology; Ddx3y and Ddx3 show 90 and 84 percent identity
at the amino acid and nucleic acid level, respectively. The differential
expression of these three genes despite their high degree of sequence
homology suggests that their differential action may be dictated by their
less homologous 5´ and 3´ untranslated sequences. The differential regulation
of expression of the three genes is currently under investigation. Genetic,
physiological, and biochemical effects of disease-causing mutations of the
luteinizing hormone receptor Leung, Bellan, Baxendale, Wu, Rennert,
Chan; in collaboration with Fechner, Steinbach, Su Constitutive activating mutations of the human
luteinizing hormone/chorionic gonadotropin receptor (hLHR) cause familial
male-limited precocious puberty (FMPP), a noncentral form of
gonadotropin-independent precocious puberty. Even though constitutive
production of testosterone, which occurs in FMPP patients, is not known to be
tumorigenic, we identified two FMPP patients who developed testicular
neoplasia. Another study of testicular tumor by Dr. Shenker’s group at The impact of an activating mutation of the
hLHR has always been considered limited to sexual development of the patient.
The abnormal social behavior of patients was thought to be secondary to
precocious sexual maturation. It is known that LHR is expressed in the brain.
We speculate that the abnormal behavior of FMPP patients is due to expression
of the mutated LHR in the brain. To test this hypothesis and study the impact
of constitutively activated LHR on spermatogenesis as well as on sexual and
neurological development, we generated a transgenic mouse strain that
expresses a fusion protein of enhanced green fluorescent protein (EGFP) to an
hLHR mutant with substitution Asp578Gly or Asp578His. Interestingly, all the
transgenic mice expressing the mutated hLHR are females. The reason for this
phenomenon is unclear. We are currently investigating the impact of the
mutated receptor on embryonic growth and development. The antithesis of FMPP is Leydig cell
hypoplasia (LCH). In LCH patients, a mutation inactivates the LHR, resulting
in reduced production of testosterone, which causes hypergonadotrophic
hypogonadism or male pseudohermaphroditism. A novel missense mutation A340T
identified in a patient with LCH results in substitution of Ile-114 by Phe,
which affects one of the leucine-rich repeats (LRRs) in the extracellular
domain of the hLHR. The mutant receptor fails to trigger cAMP production upon
hCG stimulation in transient expression studies. A fluorescence microscopy
study of the fusion protein of receptor with green fluorescent protein
revealed that the mutation does not affect trafficking of the mutated
receptor but rather affects binding of the hormone by the receptor. To study
the effect of the mutation on the conformation of the receptor, we generated
a computer model of the LRR, which clearly demonstrates the conformational
effect of the mutation and may explain the impact of mutations on the
biological activity of other proteins with LRRs. Leung MLY, Al-Muslim O,
Wu SM, Azizs A, Inam S, Awadh M, Rennert OM, Chan WY. A novel missense
homozygous inactivating mutation in the fourth transmembrane helix of the
luteinizing hormone receptor in leydig cell hypoplasia. Amer J Med Genet 2004;130A:146-153. Salameh W, Shoucair M, Guo TB, Zahed L, Wu SM,
Rennert OM, Chan WY. Leydig cell hypoplasia due to inactivation of
luteinizing hormone receptor by a novel homozygous nonsense truncation
mutation in the seventh transmembrane domain. Mol Cell Endocrinol 2004, in
press. Role
of susceptibility to thrombosis in pseudotumor cerebri of nephropathic
cystinosis Dogulu, Raygada, Chan, Rennert; in
collaboration with Gahl, Kaiser Given our recent findings regarding genetic susceptibility
to thrombosis in pseudotumor cerebri (PTC), we instituted a clinical protocol
to study the role of thrombosis susceptibility in the development of PTC in
nephropathic cystinosis patients. We are screening nephropathic cystinosis
patients who develop PTC and control nephropathic cystinosis patients without
PTC by using a thrombosis susceptibility screening panel: thrombin time (TT),
activated partial thromboplastin (APT), activated protein C resistance, serum
levels of protein C, protein S, antithrombin III, fibrinogen, factor VIII,
factor IX, factor XI, total homocysteine, antiphospholipid antibodies (ACA
and Lupus AC), Factor V Leiden mutation, Factor V G1628A polymorphism, Factor
V R2 allele, Prothrombin 20210 mutation, and 5,10-methylenetetrahydrofolate
reductase (MTHFR) gene C677T polymorphism. To date, we have recruited two patients with
pseudotumor cerebri with pre-existing nephropathic cystinosis. The thrombosis
screening panel revealed shortened TT in both patients. Thrombin time measures
the rate of fibrin monomer polymerization and is the most sensitive screening
test for decreases or abnormalities in fibrinogen. The shortened TT
demonstrates an acceleration of fibrin monomer polymerization leading to a
thrombotic tendency. Dogulu CF, Kansu T, Leung
MYK, Baxendale V, Wu SM, Ozguc M, Chan WY, Rennert OM. Evidence for genetic
susceptibility to thrombosis in idiopathic intracranial hypertension. Thromb Res 2003;111:389-395. Dogulu CF, Tsilou E, Rubin B, FitzGibbon EJ,
Kaiser MI, Rennert OM, Gahl WA. Idiopathic intracranial hypertension in
cystinosis. J Pediat
2004;145:673-678. Method
evolved for recognition of thrombophilia (MERT) Dogulu, Chan, Rennert Venous thrombosis affects one in every 1,000
individuals annually and is one of the leading causes of mortality and
morbidity, resulting in approximately 300,000 hospitalizations and 50,000
fatalities a year in the Clinical
protocol on the studies of pediatric patients with genetic and metabolic
disorders Raygada, Dogulu, Rennert; in
collaboration with Kaler, Stratakis We have instituted a clinical protocol that
provides care for patients with a variety of rare genetic disorders, offers an
opportunity for training in clinical genetics, dysmorphology, and metabolic
genetics, and serves to spearhead the development of new research protocols
on particular aspects of diagnosis and treatment of specific genetic
diseases. We evaluate patients with a broad spectrum of metabolic and genetic
conditions, offer genetic counseling services to patients and their families
to assess risk, and provide information on preventive measures and testing
options. The disorders we study include chromosomal and Mendelian disorders
of childhood and/or adults, congenital anomalies and/or birth defects,
dysmorphic syndromes, familial cancer syndromes, multifactorial disorders,
and metabolic abnormalities. Patients and/or family members with genetic
disorders may provide DNA for storage and/or testing. The overall purpose of
the protocol is to support NICHD’s training and research missions by
expanding the spectrum of diseases that can be seen in our clinics and wards
and to recruit a diverse population of patients and/or biological samples to
provide NICHD investigators and trainees with hands-on experience related to
diagnosis, management, follow-up, treatment, and genetic counseling. The
protocol also provides an opportunity to evaluate patients with unusual or
challenging genetic and metabolic disorders who may not be eligible for an
existing research protocol; often it is not possible to determine protocol
eligibility without prospective evaluation conducted at the NIH. Such
patients may be of exceptional educational value for clinical staff at all
levels, and their evaluation may catalyze the recognition of new disease
processes and need for research initiatives. Furthermore, the evaluation of
such challenging patients is necessary to sustain the analytic and innovative
faculties of clinical research staff at all levels, from student to clinical
fellow to senior staff member. Ifon ET, Pang ALY, Johnson W, Cashman K,
Zimmerman S, Muralidhar S, Chan WY, Casey J, Rosenthal LJ. U94 alters FN1 and
ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell
line PC3. Cancer Cell Internat 2004,
in press. Ohta S, Lai EW, Pang ALY, Brouwers FM, Chan
WY, Eisenhofer G, de Krijger R, Ksinantova L, Blazicek P, Breza J, Kvetnansky
R, Wesley RA, Pacak K. Down-regulation of metastasis suppressor gene in
malignant pheochromocytoma. Internat J
Cancer 2004, Nov 2; [Epub ahead of
print]. Raygada M, Rennert OM. Congenital generalized
lipodystrophy: profile of the disease and gender differences in two siblings.
Clin Genet 2004, in
press. Roth J, Raygada M, Devaskar S,
Montrose-Rafizadeh C, LeRoith D. Insulin and the brain. In: Adelman G, Smith
BH, eds. Encyclopedia of Neuroscience,
3rd edition, [on CD Rom], Oxford, UK: Elsevier; 2003. Stratakis C, Rennert OM. Turner syndrome: an
update. The Endocrinologist 2004, in press. COLLABORATORS Martin Dym, PhD, Patricia Fechner, MD, William Gahl, MD, PhD, Clinical Director, NHGRI, Muriel I. Kaiser, MD, Ophthalmic Genetics and Visual Function
Branch, NEI, Stephen Kaler, MD, MPH, Clinical Director, NICHD, Chris Y.F. Lau, PhD, Malcolm M. Martin, MD, Neelakanta Ravindranath, PhD,
Center for Scientific Review, NIH, Peter Steinbach, PhD, Center for Information Technology, NIH, Timothy Stitely, MS, Unit on Computer Support Services, NICHD, Constantine Stratakis, MD,
DSc, Heritable Disorders Branch, NICHD,
For
further information, contact chanwy@mail.nih.gov |
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