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128
CHAPTER 14
of the cells and CD3
T lymphocytes making up 2% of the cells. These percentages would be con-
sistent with DiGeorge syndrome where the patient basically has B lymphocytes but no T lympho-
cytes.
Figure 14-14E Two-dimensional dot plot of adenosine deaminase deficiency (ADA; “Bubble
Boy”). Peripheral blood sample from a patient is run through flow cytometry using two fluores-
cent-labeled antibodies: CD20 antibody which labels all B lymphocytes and CD3 antibody which
labels all T lymphocytes. The two-dimensional dot plot shows four populations of cells with CD
20
B lymphocytes making up only 2% of the cells and CD3
T lymphocytes making up only 2%
of the cells. These percentages would be consistent with ADA (“Bubble Boy”) where the patient
basically has no B lymphocytes and T lymphocytes.
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Identification of Human Disease Genes
129
There is no one particular way that a human gene involved in a dis-
ease is identified. Molecular biologists use a large variety of molecular techniques to inves-
tigate each situation on a case-by-case basis.
Identification of a Human Disease Gene Through a Chromosome
Abnormality
A. SOTOS SYNDROME (also called Cerebral Gigantism; Figure 15-1). Sotos syndrome
is a syndrome found in infants that tend to be large at birth and continue to grow rap-
idly during the early years of childhood. Clinical findings include macrocephaly, high
forehead, frontal bossing, downward slanting of palpebral fissures, hypertelorism,
prominent jaw, sparse hair in frontoparietal region, high arched palate, mental retarda-
tion, and poor coordination.
B. A Sotos patient was found with a chromosome
translocation between band q35 on chromo-
some 5 and band q24 on chromosome 8, t(5;8)
(q35;q24).
C. The DNA from this patient was isolated and
inserted into various cloning vectors (e.g., cos-
mids, bacterial artificial chromosomes, and
Plasmid artificial chromosomes).
D. Clones containing the breakpoint were identi-
fied by FISH (fluorescence in situ hybridiza-
tion) and the DNA sequencing around the
breakpoint was done.
E.
The DNA sequence was found to be a homologue of the mouse Nsd1 gene by com-
puter analysis of gene databases.
F.
Subsequently, a human genomic library was screened using a DNA probe constructed
from the mouse Nsd1 gene, and the human NSD1 gene on chromosome 5q35 was
cloned.
G. Once the NSD1 gene was cloned, DNA from other Sotos patients was analyzed and mu-
tations and/or microdeletions in the NSD1 gene were found.
H. This confirmed that mutations and/or microdeletions in the NSD1 gene are directly in-
volved in the Sotos syndrome. The NSD1 gene encodes for the NSD1 protein (nuclear
receptor binding SET domain protein 1) which is a histone methyltransferase that
acts as a transcriptional intermediary factor capable of negatively and positively influ-
encing transcription. Figure 15-1 shows a young girl with Sotos syndrome.
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130
CHAPTER 15
Identification of a Human Disease Gene Through Pure Transcript Mapping
A. TREACHER COLLINS FRANCESCHETTI
SYNDROME (TCOF; Figure 15-2). A First
Arch syndrome results from abnormal devel-
opment of pharyngeal arch 1 and produces
various facial anomalies. First Arch syndromes
are caused by a lack of migration of neural
crest cells into pharyngeal arch 1. A well-
described First Arch syndrome is the TCOF syn-
drome. TCOF is an autosomal dominant genetic
disorder caused by a mutation in the TCOFI
gene on chromosome 5q32–33.1 for the treacle
protein. Clinical findings include hypoplasia of
the zygomatic bones and mandible resulting in
midface hypoplasia, micrognathia, and retrog-
nathia; external ear abnormalities including
small, absent, malformed, or rotated ears; and
lower eyelid abnormalities including coloboma.
● Figure 15-2 Treacher Collins France-
schetti Syndrome.
B. Genetic linkage studies established linkage to markers on chromosome 5q32–33.1.
The purpose of genetic linkage is to identify a crude chromosomal location of a partic-
ular gene locus or gene allele. Genetic loci on the same chromosome that are physi-
cally close to one another tend to stay together during meiosis in germ cells and are
thus genetically linked. Recombination occurs in germ cells during meiosis. As re-
combination occurs, if there is a large physical distance between two gene loci, there is
a good chance that crossover will occur between them and the two gene loci will not
stay together and therefore segregate independently among the gametes. However, if
there is a small physical distance between two gene loci, there is a good chance that
crossover will not occur between them and the two gene loci will stay together and
therefore segregate together among the gametes. In genetic linkage studies, disease
genes are mapped by measuring recombination against a panel of different markers
spread over the entire genome.
C. A YAC clone contig for chromosome 5q32–33.1 was constructed. A number of genes
were identified in this region. A clone contig is one of a set of overlapping clones that
represent a continuous region of DNA. More genetic linkage studies eventually pro-
duced a candidate gene called the TCOF1 gene.
D. The TCOF1 gene showed no homologous relationship to any other gene by computer
analysis of gene databases.
E.
DNA from other TCOF patients was analyzed and mutations and/or microdeletions in
the TCOF1 gene were found.
F.
This confirmed that mutations and/or microdeletions in the human TCOF1 gene are
directly involved in the TCOF syndrome. The TCOF1 gene encodes for the treacle pro-
tein which is a nucleolar protein related to the nucleolar phosphoprotein Nopp140
both of which contain LIS1 motifs leading to the speculation of microtubule dynam-
ics involvement. In addition, treacle protein interacts with the small nucleolar ribonu-
cleoprotein hNop56p leading to the speculation of ribosomal biogenesis involvement.
Figure 15-2 shows a young boy with TCOF syndrome.
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131
IDENTIFICATION OF HUMAN DISEASE GENES
Identification of a Human Disease Gene Through Large Scale DNA
Sequencing
● Figure 15-3 Branchio-Oto-Renal Syn-
drome.
B. A BOR patient was found with a rearrangement of chromosome 8q13.
C. A PAC clone contig for a region around 8q13 was constructed.
D. Large scale DNA sequencing of the 8q13 region was done.
E.
The DNA sequence was found to be a homologue of the Drosophila Eya gene (eyes
absent) by computer analysis of gene databases.
F.
Subsequently, a cDNA library using 9-week total fetal mRNA was screened using a DNA
probe constructed from the Drosophila Eya gene, and the human EYA1 gene was
cloned.
G. Once the EYA1 gene was cloned, DNA from other BOR patients was analyzed and mu-
tations and/or microdeletions in the EYA1 gene were found. This confirmed that muta-
tions and/or microdeletions in the human EYA1 gene are directly involved in the BOR
syndrome. The EYA1 gene encodes for the EYA1 protein (eyes absent homolog 1 pro-
tein) which has intrinsic phosphatase activity enabling it to serve as a promoter-
specific transcriptional coactivator. EYA1 protein interacts with several other proteins,
including SIX1 and SIX5, to regulate the activity of genes involved in many aspects of
embryonic development. Figure 15-3 shows a young girl with an ear malformation typ-
ical of BOR syndrome.
A. BRANCHIO-OTO-RENAL SYNDROME (BOR
SYNDROME; Figure 15-3). External ear
malformations and renal anomalies occur in
several multiple congenital anomaly syn-
dromes (e.g., BOR syndrome, CHARGE asso-
ciation, Townes-Brocks syndrome, Nager
syndrome, Miller syndrome, and diabetic em-
bryopathy). The BOR syndrome is an autoso-
mal dominant genetic disorder found in in-
fants. Clinical findings include pharyngeal
fistulas along the side of the neck, malforma-
tion of the external and internal ear, hearing
loss, hypoplasia, or absence of the kidneys.
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132
CHAPTER 15
Identification of a Human Disease Gene Through Comparison of Human
and Mouse Maps
A. WAARDENBURG SYNDROME TYPE 1
(WS1; Figure 15-4). WS1 is an autosomal
dominant genetic disorder caused by muta-
tion in the PAX3 gene on chromosome 2q35
for the PAX3 paired box protein. The PAX
genes are characterized by a 128 amino acid
DNA-binding domain called a paired box.
Clinical findings include dystopia cantho-
rum (lateral displacement of the inner can-
thi), growing together of eyebrows, lateral
displacement of lacrimal puncta, a broad
nasal root, heterochromia of the iris, con-
genital deafness or hearing impairment, and
piebaldism including a white forelock and a
triangular area of hypopigmentation.
B. Genetic linkage studies established linkage to
markers on human chromosome 2q. The human chromosome 2q region has strong
synteny (i.e., general correspondence) to a portion of mouse chromosome 1.
C. A mouse mutant called the Splotch (Sp) mutant was described with pigmentary ab-
normalities due to the patchy absence of melanocytes. The phenotypic similarities
between WS Type 1 and the Splotch mutant mouse suggested that homologous genes
were involved. The mouse Pax-3 gene was linked to the Splotch mutant.
D. Subsequently, a human genomic library was screened using a DNA probe constructed
from the mouse Pax-3 gene, and the human PAX3 gene was cloned.
E.
Once the PAX3 gene was cloned, DNA from other WS Type 1 patients was analyzed,
and mutations and/or microdeletions in the PAX3 gene were found. This confirmed that
mutations and/or microdeletions in the human PAX3 gene are directly involved in the
WS Type 1 syndrome. The PAX3 gene encodes for the PAX3 protein (paired box pro-
tein 3) which is a DNA-binding transcription factor that is expressed in the early em-
bryo and regulates neural crest-derived cell types, including melanocytes. Figure 15-4
shows a teenage boy with Waardenburg syndrome.
● Figure 15-4 Waardenburg Syndrome
Type 1.
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