Reproductive Genetics

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Chapter 5 Reproductive Genetics

INTRODUCTION

We learn quickly in life that variability exists between individuals. For many of us the differences are as simple as hair or eye color. For others, the differences are profound and can take the form of a severe birth defect or syndrome. As a whole, these differences add up and cause significant morbidity and mortality. A Canadian study has found that approximately 12% of individuals suffer health problems related to or caused by genetic disease from birth to early adulthood.1 Genetics is the study of traits and inherited differences between individuals; as Gregor Mendel demonstrated, we were able to learn the principles of inheritance without knowing about or understanding DNA and its organization in the genome. The traditional study of medicine and medical genetics is, however, about to be revolutionized. In this technological revolution of genomic medicine, we see that a fundamental understanding of genetics and the principles of inheritance are still required.

The practice of clinical medical genetics has been significantly transformed over the past 15 years, from a somewhat arcane specialty dealing with prenatal diagnosis, chromosome abnormalities, and dysmorphology to a challenging and cutting edge specialty taking the lead in introducing genomic medicine to the broader field of medicine. In 2001, the Human Genome Project announced that a rough sequence of the human genome had been completed.2,3 In its original iteration the Human Genome Project was an attempt to sequence and determine the linear sequence of 3 billion base pairs and map the individual genes encoded by this sequence. Genomics is the study of our complete set of genes, their functions and interactions with themselves and their environment, and how genetic variation contributes to disease risk and response to treatment (i.e., pharmacogenomics). This sequencing and mapping effort has now spawned functional genomics, which seeks to identify gene function, regulation, and gene interaction.

The knowledge and technologies learned from this effort have, and will have, as profound an impact on the field of reproductive medicine as any other. The physician in obstetrics and gynecology will not only have to have an understanding of genetics and the principles of inheritance, but he or she will also have to understand genomics and its derivative technologies and how these will affect the risk, diagnosis, and treatment of medical disorders. Physicians will not only be practicing the traditional paradigm of diagnosis and treatment of disease, but will also be recognizing and treating genomically derived disease risk prior to disease manifestation. They must be ready to deal with all the ethical, legal, and social implications of this revolution. Although the era of genomic medicine will bring new tools to individualize disease risk, emphasize prevention, and treat disease, the simple basic recording and understanding of the family history will still be a fundamental component of this new era.4

THE HUMAN GENOME

The Human Genome Project has resulted in some surprising revelations.2,3,5 Less than 2% of the human genome has genes that code for proteins. This was a surprising finding because it was thought that the human genome would reflect the complexity of human development and it was estimated that the human genome would contain up to 120,000 genes. The first report of the Human Genome Project estimated that the human genome contained only 30,000 to 35,000 genes. Drosophila melanogaster had been found to have 14,000 genes and the mustard plant Arabidopsis thaliana 26,000 genes.6,7 It is likely now that the complexity of human growth and development and its relation to disease is not going to be explained by just new gene discovery. The previous dogma that one gene produces one protein has now been replaced by the theory of alternative splicing, in which a gene’s exons or coding regions are shuffled to produce alternative forms of a related protein (Fig. 5-1).

Exons are sequences of a gene that are transcribed into a protein. Introns are regions of a gene that do not code for a protein. When the mRNA of a gene is translated, exons are spliced together. Exons have been found to comprise only 1% of the human genome and introns about 25%. Typically more than 25 times the amount of DNA in a gene is not associated with protein structure and function. This has significant implications for DNA testing and interpretation of a test’s sensitivity and specificity. Regions of genes on chromosomes have been described as existing in gene-rich oases separated by gene-sparse deserts.

Over 10% of the genome is composed of repeated sequences of DNA that may be related to chromosome structure. Long interspersed repetitive elements and short interspersed repetitive elements have been described, including Alu repeat sequences. Alu sequences have been found in gene-rich regions and may play a role in genetic recombination.

GENES

With the recent advances of the Human Genome Project and the rapid adoption of these findings into clinical medicine, the clinician will need to understand the organization of the human genome. The average size of a gene is about 3000 base pairs. Chromosome 1, the largest chromosome in a karyotype, has about 2968 genes; the Y chromosome, the smallest, has 231 genes. Genes are distributed in random areas of the chromosome, with some chromosomes gene-rich and some gene-poor. Chromosomes 17, 19, and 22 are gene-dense. Chromosomes 4, 8, 13, 18, and Y are gene-sparse. It is not surprising then that chromosomes 13, 18, and 21, which are the common trisomies that can survive to birth, have the fewest genes.

Genes can be altered and cause human disease through several mechanisms. Point mutations, in which there is a single change of a DNA base in the sequence, can have multiple effects on gene function. Missense mutation is the substitution of a single base in the DNA sequence, incorporating a different amino acid in the protein sequence. These mutations may have little effect on protein function if the amino acid substituted is similar to the original one. If the substitution incorporates a very different amino acid, the protein structure and function may be significantly affected. A silent mutation is the substitution of a single base in the DNA sequence that does not change the specific amino acid in the sequence. Nonsense mutations are the substitution of a single DNA base in the sequence, causing the premature termination or truncation of a protein. In addition to point mutations, frame-shift mutations can also lead to an altered or truncated protein. Frame-shift mutations are deletions or additions of DNA bases that are not multiples of three. These changes lead to a downstream change in the reading frame, often truncating a protein. In general, these types of mutations result in loss of function of a protein and alter the phenotype by decreasing the activity of a protein.

CHROMOSOME ABNORMALITIES

Chromosome abnormalities fall into two general categories: numerical and structural. In numerical abnormalities the total number varies from the normal, 46. With structural abnormalities, the chromosome structure has been physically rearranged.

Numerical Abnormalities

Abnormalities of chromosome number are the most commonly recognized clinical chromosome abnormalities. Structural chromosome anomalies contribute significantly to birth defects, infertility, and recurrent pregnancy loss. Abnormalities in chromosome number generally arise from mistakes at cell division where there is either gain or loss, or both, of chromosomes in daughter cells, or nondisjunction. Nondisjunction can occur either during meiosis or mitosis. Cells resulting from this occurrence are aneuploid because their chromosome number is not a multiple of the haploid number 23. Nondisjunction can occur during either meiosis I or meiosis II. Either can produce an aneuploid conception (Fig. 5-3). Nondisjunction or anaphase lag can occur at mitosis, causing mosaicism, the presence of two or more cell lines in an individual. Mosaicism is often seen with the sex chromosome abnormality Turner’s syndrome, where up to 50% of cases have some form of mosaicism.9 The common numerical chromosome abnormalities are listed in Table 5-1. Numerical chromosome abnormalities can also involve multiples of the haploid number of 23 chromosomes. Triploidy, with 69 chromosomes, usually arises from the fertilization of a single egg by two sperm. Triploid conceptions are seen in about 15% of chromosomally abnormal miscarriages and occasionally survive to term. Tetraploidy has a modal number of 92 chromosomes and occurs in a small percentage of spontaneous losses.

Table 5-1 Chromosome Abnormalities

Chromosome Clinical Findings
Trisomy 13 Severe CNS abnormalities, holoprosencephaly, microphthalmia, coloboma, cleft lip and palate, abnormal auricles, polydactyly, cardiac defects
Trisomy 18 CNS malformations, prominent occiput, micrognathia, small mouth, low-set ears, hypoplastic nails, overlapping fingers, cardiac defects
Trisomy 21 Microcephaly, flat occiput, Brushfield spots, epicanthal folds, simian crease, conotruncal cardiac defects, redundant skin on nape of neck, hypotonia
XXX Normal female phenotype, tall stature, may have some learning and developmental disabilities, normal fertility
45,X Turner’s Short stature, gonadal failure, absent secondary sex characteristics, webbed neck, low-set hairline, coarctation of the aorta, horseshoe kidney, may have some spatial learning disabilities
XXY Klinefelter’s May be tall, infertile, small testis, learning disabilities, gynecomastia

Structural Chromosome Abnormalities

Structural chromosomal rearrangements occur when chromosomes break and the original architecture is not restored. Chromosome rearrangements are balanced when the diploid genetic state is maintained. When rearrangements are unbalanced, they result in aneuploidy for one or more chromosome segments. These structural rearrangements may segregate and be termed familial, or they may occur as a first or new event, de novo. Balanced familial chromosome rearrangements are in most cases truly balanced and represent little risk of birth defects or mental retardation. De novo chromosome rearrangements that appear balanced carry a small risk of aneuploidy at the molecular level of about 5% for birth defects and developmental delay.

Translocations involve the exchange of chromosome arms between two different chromosomes. Reciprocal translocations occur when there is breakage within two arms and reciprocal exchange of the distal segments, creating a derivative chromosome (Fig. 5-4). In most cases balanced translocation carriers are phenotypically normal but are at risk for producing unbalanced gametes during gametogenesis. In a balanced translocation carrier the types of chromosome segregation can be complex, resulting in a normal segregation pattern, a balanced translocation pattern, and an unbalanced pattern producing a partial trisomy and partial monosomy for the chromosomes involved (see Fig. 5-4).

When the short arms of two acrocentric chromosomes are involved in a translocation, the long arms are joined in the centromeric region of one chromosome, with the loss of the short arms of the acrocentric chromosomes producing Robertsonian translocations. Because the short arms of acrocentric chromosomes contain redundant ribosomal genetic material, the loss of this material is of no phenotypic consequence. As with balanced translocations, the products of meiotic segregation can be either balanced or unbalanced (Fig. 5-5).

Other structural abnormalities can produce pregnancy loss and birth defects. When two breaks occur in a single chromosome with the interstitial segment flipped 180 degrees at the time of repair, an inversion can occur. If this involves each arm of the chromosome a pericentric inversion is produced. If only a single arm of the chromosome is involved in the inversion, a paracentric inversion is produced (Fig. 5-6). Each has unique and different implications for gamete production and pregnancy loss. With chromosome duplication a chromosome segment of varying size can be duplicated, causing a partial trisomy for this segment. With a deletion, a segment of varying size is missing, causing a genetic imbalance or partial monosomy. This condition, in which a second copy of a gene or segment of chromosome is missing, resulting in an abnormal phenotype or clinical presentation, is known as haploinsufficiency.

Fluorescent In Situ Hybridization

Traditional cytogenetics has always been limited by the band level or resolution of the karyotype. Even with high-resolution banding that could elongate chromosomes and resolve an increasing number of bands per chromosome, one could never be certain that a chromosome was intact at the molecular level. With advances in DNA technology and cytogenetics it is now possible using fluorescent in situ hybridization (FISH) to analyze chromosomes at the molecular level for changes in the DNA. Molecular cytogenetics has now revolutionized cytogenetics by permitting (1) the analysis of DNA structure within a chromosome down to within 10 to 100 kb and (2) the diagnostic analysis of nondividing interphase cells, producing a significant impact on the field of prenatal diagnosis and that of preimplantation genetic diagnosis.10

FISH technology uses DNA probes that can bind or anneal to specific DNA sequences within the chromosome. A denatured probe is incubated with native DNA from a cell that has also been denatured to the single-strand state. The probe substitutes biotin-dUTP or digoxigenin-UTP for thymidine. After the probe has annealed to native DNA, the probe–DNA complex can be detected by adding fluorochrome-tagged avidin that binds to biotin or fluorochrome-labeled antidigoxigenin. This signal can be additionally amplified by adding antiavidin and the complex visualized by fluorescence microscopy. Using several different fluorochromes tagged to different DNA probes, different chromosomes or chromosome segments can be simultaneously visualized within a cell as different colored signals. The ability to detect specific gene segments that are either present or missing has permitted the diagnosis of contiguous gene syndromes at the DNA level as well as translocations in interphase nuclei, often in single cells.

Material for FISH can be either metaphase chromosomes obtained from dividing cells or interphase nuclei from cells that are not dividing. Slides are pretreated with RNAse and proteinase to remove RNA that may cross-hybridize with the probe and chromatin. The slides are heated in formamide to denature the DNA and then fixed in cold ethanol. The probe is then prepared for hybridization by heating. The probe and chromosome preparation are then mixed and sealed with a coverslip at 37°C for hybridization. By varying the incubation temperature or the salt composition of the hybridization solution, the stringency of the binding can be increased and the background labeling reduced.

Applications of FISH

The technique of in situ hybridization first proved useful for localizing genes to chromosomes. With the introduction of fluorescence labeling, in situ hybridization proved invaluable in identifying chromosome abnormalities that could not be identified by traditional banding methods. FISH also played a key role in one of the most unusual discoveries of modern genetics, that of genomic imprinting.

FISH technology has been developed in three forms. Centromeric or alpha-satellite probes are relatively chromosome specific and have had probably the broadest application in interphase genetics.11,12 These probes produce somewhat diffuse signals near the centromere with adequate strength, but do not cross-hybridize with chromosomes that have similar centromeric sequences. Single copy probes have now been developed that give discrete signals from a specific band on a chromosome and avoid the issue of cross-hybridization. These can also be used to detect copy number and specific chromosome regions known to be associated with syndromes. Single copy probes and centromeric probes for chromosomes 13, 18, 21, X, and Y have been developed for use in prenatal diagnosis. It is also possible to “paint” whole chromosomes using FISH. Using spectral karyotyping technology that combines mixtures of fluorochromes, it is now possible to produce a unique fluorescent pattern for each individual chromosome in 24 different colors. This technology permits the detection of complex chromosome rearrangements that cannot be seen with traditional cytogenetic techniques (Fig. 5-7).

Contiguous Gene Syndromes

Contiguous gene syndromes are also known as microdeletion syndromes, or segmental aneusomy.10 These are deletions of a contiguous stretch of chromosome that usually involve multiple genes. The contiguous gene syndromes were first described in 1986 using classical cytogenetic methodologies. Using FISH, submicroscopic deletions can now be identified at the level of DNA; this has permitted the characterization of the smallest deleted region consistently associated with a syndrome, known as the critical region. By identifying the critical region of a syndrome, it is often possible to identify the specific genes that, when missing, are associated with the syndrome (Fig. 5-8). A recent compendium of deletion syndromes has reported 18 deletion and microdeletion syndromes spread over 14 chromosomes.13 Some of the more common deletion and mirodeletion syndromes and their clinical findings are shown in Table 5-2.

Table 5-2 Deletion and Contiguous Gene Syndromes

Deletion Syndrome Clinical Description
4p– Wolf-Hirschhorn Microcephaly, hypertelorism, frontal bossing, cleft lip and palate, “Greek helmet” face, mental retardation, hypotonia, cardiac anomalies
5p– Cri du chat Microcephaly, characteristic “cat cry,” hypotonia, mental retardation
7q11.23 Williams Round face, full lips, stellate iris, supravalvular aortic stenosis, mental delay, “cocktail personality”
11p13 WAGR Wilm’s tumor, aniridia, genital abnormalities, mental retardation
15q11-13 Angelman Blonde hair, prognathism, seizures, ataxia, laughter, hypotonia, mental retardation
15q11-13 Prader-Willi Birth hypotonia, hyperphagia, obesity, short stature, hypogonadism, mental retardation
16p13 Rubinstein-Taybi Characteristic facies, beaked nose, microcephaly, mental retardation
17p11 Smith-Magenis Brachycephaly, prominent chin, short stature, mental retardation, behavioral phenotype
17p13 Miller-Dieker Microcephaly, lissencephaly, growth retardation, seizures, mental retardation
20p12 Alagille Cholestasis, heart defects, ocular findings, skeletal defects
22q11 DiGeorge/CATCH 22 Thymic and parathyroid hypoplasia, calcium abnormalities, conotruncal heart defects, short stature, behavioral and learning problems

GENERAL PRINCIPLES OF MENDELIAN GENETICS