Sex Linkage and Extranuclear Inheritance

Mendel's peas gave the same results no matter which parent carried which allele, but many real traits refuse to behave that way. A son inherits color blindness from his mother, and a deletion on chromosome 15 causes one disease when it comes from the father and another when it comes from the mother. This chapter explains why the sex of the parent, and the sex of the child, can change the outcome of a cross.

Section 1Sex determination systems

In most animals and in a minority of flowering plants, individuals come in two sexes that make different gametes: females make large, nutrient rich eggs and males make small, mobile sperm or pollen. Sex determination is the mechanism that decides which developmental path an embryo takes. It matters here because when sex is chromosomal, the chromosomes that decide sex also carry ordinary genes whose inheritance is tied to sex.

Chromosomal sex determination

In chromosomal systems, the two sexes differ in their sex chromosomes, and every other chromosome is an autosome. The sex that carries two different sex chromosomes makes two kinds of gametes and is called the heterogametic sex; the sex with two matching sex chromosomes makes one kind of gamete and is the homogametic sex.

• XX/XY. Females are XX (homogametic) and males are XY (heterogametic). This is the system of mammals, of Drosophila and of a few dioecious plants such as white campion. Because each sperm carries either an X or a Y, the father's gamete decides the sex of each child, and the expected sex ratio is 1:1.
• XX/XO. Females are XX and males have a single X and no partner chromosome (written XO, where O means absence). Many grasshoppers and crickets use this system. Males make two kinds of sperm, those with an X and those with no sex chromosome. The nematode Caenorhabditis elegans is a variant: XX individuals are self fertilizing hermaphrodites and XO individuals are males.
• ZZ/ZW. Here the female is heterogametic (ZW) and the male is homogametic (ZZ). Birds, butterflies and moths, and some fish and reptiles use it. The letters Z and W are used precisely so that no one confuses this system with XX/XY. In birds, the egg (not the sperm) decides the sex of the offspring. Evidence suggests that two doses of the Z linked gene DMRT1 push the bird gonad toward a testis.

Haplodiploidy

Bees, wasps and ants (the order Hymenoptera) have no sex chromosomes at all. Fertilized eggs develop into diploid females and unfertilized eggs develop into haploid males, a system called haplodiploidy. A queen honeybee controls whether each egg she lays is fertilized, and so its sex. Because a male is haploid, he makes sperm by mitosis, and every sperm he produces is genetically identical to every other.

In many hymenopterans the real switch is a single locus called complementary sex determiner (csd). Individuals heterozygous at csd become female; individuals that are hemizygous (haploid) become male. Inbreeding produces some eggs homozygous at csd, which develop as sterile diploid males.

Mammals: SRY decides

The study of people with unusual numbers of sex chromosomes showed that in humans it is the presence of a Y chromosome, not the number of X chromosomes, that makes an embryo male. A person with 45,X (one X, no second sex chromosome) has Turner syndrome and develops as a female, usually with short stature and ovaries that fail early. A person with 47,XXY has Klinefelter syndrome and develops as a male, typically tall with small testes and reduced fertility. People with 47,XXX are female and those with 47,XYY are male.

The critical gene on the Y is SRY (sex determining region Y), located on the short arm near the boundary of the pseudoautosomal region. SRY encodes a transcription factor with an HMG box, a DNA binding domain that bends DNA. In the bipotential gonad of a six to seven week embryo, SRY switches on the autosomal gene SOX9. SOX9 makes gonad cells become Sertoli cells, which secrete anti Müllerian hormone to remove the female duct system, while Leydig cells make testosterone to masculinize the rest of the body. Without SRY, the gonad follows the ovarian pathway, supported by genes such as WNT4, RSPO1 and FOXL2.

Two rare conditions confirm SRY's role. In XX males, an abnormal crossover during the father's meiosis has carried SRY from the Y onto the X, so the child has two X chromosomes and testes. In XY females (Swyer syndrome in some cases), the Y is present but SRY is deleted or carries a loss of function mutation, and the gonads develop as nonfunctional streaks.

The pseudoautosomal regions

The human X and Y are very different in size and gene content: the X carries roughly 800 to 900 protein coding genes while the Y carries only a few dozen. Yet in male meiosis the X and Y must pair and segregate. They do so through short regions of shared sequence at their tips, the pseudoautosomal regions (PAR1 at the end of the short arms and PAR2 at the end of the long arms). In male meiosis at least one crossover occurs in PAR1, which is why genes there, such as SHOX, are inherited as though they were autosomal. Outside the PARs, the male specific region of the Y does not recombine and passes intact from father to son.

Drosophila: the X to autosome ratio

Fruit flies have XX females and XY males, but their sex is decided differently. In flies the Y carries no sex determining gene; instead, sex depends on the ratio of X chromosomes to sets of autosomes, the X:A ratio. Calvin Bridges worked this out in the 1920s by studying flies with unusual chromosome sets. A ratio of 1.0 or higher produces a female, 0.5 or lower produces a male, and values in between produce intersexes. Molecularly, a double dose of X linked signal genes switches on Sex lethal (Sxl), which starts a cascade of alternative RNA splicing (Sxl to transformer to doublesex) that produces female development. The Y is still needed in male flies for fertility, because it carries genes for sperm motility, but an XO fly is a sterile male and an XXY fly is a fertile female.

Environmental sex determination

In some animals, sex is not decided by genotype at all. Many turtles, all crocodilians and some lizards show temperature dependent sex determination (TSD): the temperature at which an egg incubates during a sensitive window of development decides the sex. In many turtles warm nests produce females and cool nests produce males; in the American alligator the pattern is reversed, with cool incubation giving females. Temperature acts partly through aromatase, the enzyme that converts testosterone to estrogen. Some fish, such as clownfish, even change sex as adults in response to social cues.

Section 2X linked inheritance and pedigrees

Genes on the X chromosome are X linked. A male has one X, so for X linked genes he is neither homozygous nor heterozygous but hemizygous: whatever allele his single X carries is expressed. A female has two X chromosomes and can be homozygous or heterozygous, just as for autosomal genes. This asymmetry gives X linked traits their signature: results that change when the sexes of the parents are swapped.

Morgan's white eyed fly

In 1910 Thomas Hunt Morgan found a single male fruit fly with white eyes in a bottle of normal, red eyed flies. He crossed it to a red eyed female. All the F1 flies had red eyes, so white was recessive. When he intercrossed the F1, the F2 contained 3 red to 1 white, a Mendelian ratio, but with a twist: every white eyed fly was male. Half the F2 males had white eyes and none of the females did.

Morgan proposed that the gene lies on the X and that the Y has no copy. Using the convention X⁺ for the wild type (red) allele and X^w for white, the original male was X^wY and the female X⁺X⁺. The F1 females were X⁺X^w and the F1 males X⁺Y. In the F2, daughters received X⁺ from their father and so were all red eyed, while sons received an X from their heterozygous mother and were X⁺Y or X^wY in equal numbers.

The decisive test was the reciprocal cross: a white eyed female (X^wX^w) crossed to a red eyed male (X⁺Y). For an autosomal gene the reciprocal cross gives the same result as the original. Here it did not. All F1 daughters were red eyed (X⁺X^w) and all F1 sons were white eyed (X^wY). This pattern, in which sons resemble their mothers and daughters resemble their fathers, is called crisscross inheritance. Intercrossing these F1 flies gives an F2 of 1 red female : 1 white female : 1 red male : 1 white male.

Bridges and nondisjunction: proof of the chromosome theory

Morgan's student Calvin Bridges pushed the argument to its conclusion. When he crossed white eyed females to red eyed males, about 1 in 2,000 offspring broke the crisscross rule: a few daughters were white eyed and a few sons were red eyed. Bridges predicted that these exceptions came from nondisjunction, the failure of the two X chromosomes to separate in the mother's meiosis. An egg carrying both X^w chromosomes, fertilized by a Y bearing sperm, would give an X^wX^wY fly: female (X:A = 1.0) and white eyed. An egg with no X, fertilized by an X⁺ sperm, would give an X⁺O fly: a red eyed, sterile male. Under the microscope, the exceptional females had a Y and the exceptional males lacked one, exactly as predicted: the most convincing proof yet that genes are carried on chromosomes.

Reading X linked recessive pedigrees

In human families we cannot set up crosses, so we read pedigrees. An X linked recessive trait leaves a characteristic set of fingerprints:

• Many more males than females are affected. A male needs only one copy of the allele; a female needs two. If the allele frequency is q, the expected frequency of affected males is q and of affected females is q². For red green color vision defects, which affect about 8 percent of males of northern European ancestry, the expected frequency in females is about 0.08² = 0.0064, or roughly 0.6 percent, close to what is observed.
• No male to male transmission. A father gives his X only to daughters and his Y to sons, so an affected father cannot pass the trait to his sons.
• All daughters of an affected male are carriers. Each daughter receives her father's only X.
• The trait often skips a generation, passing from an affected grandfather through a carrier daughter to an affected grandson.
• Sons of carrier mothers have a 1/2 chance of being affected, and daughters of carrier mothers have a 1/2 chance of being carriers.
• An affected female usually has an affected father and a mother who is a carrier or affected.

X linked dominant and Y linked patterns

An X linked dominant trait appears in anyone with at least one copy of the allele. Its pedigree features are different: an affected father passes the trait to all of his daughters and none of his sons, and an affected heterozygous mother passes it to half of her children of either sex. Because females have two chances to inherit the allele, affected females outnumber affected males, often about two to one. Examples include X linked hypophosphatemic rickets, in which a defect in the PHEX gene causes the kidney to waste phosphate, and incontinentia pigmenti and Rett syndrome, in which the mutation is usually lethal or far more severe in hemizygous males, so most patients are female.

Y linked (holandric) traits pass only from father to son, to every son, and never to daughters. Nearly all known Y linked traits concern male fertility, such as deletions that block sperm production.

Section 3Dosage compensation and X inactivation

Gene dosage matters. Extra or missing copies of an autosome are usually lethal or cause serious disease because hundreds of genes are suddenly expressed at the wrong level. Yet in XX/XY species, females have two copies of every X linked gene and males have one, with no harm to either sex. The reason is dosage compensation, a set of mechanisms that equalize the expression of X linked genes between the sexes. Remarkably, three well studied animals solve the problem in three different ways.

Barr bodies and the Lyon hypothesis

In 1949, Murray Barr and Ewart Bertram noticed a small, darkly staining mass at the edge of the nucleus in nerve cells of female cats that was missing in males. This Barr body turned out to be a condensed, transcriptionally silent X chromosome, a form of facultative heterochromatin. In 1961 Mary Lyon proposed what is now called the Lyon hypothesis:

1. In each somatic cell of a female mammal, one X chromosome is inactivated and becomes a Barr body.
2. Inactivation happens early in embryonic development.
3. In the embryo proper, the choice of which X to silence, maternal or paternal, is random and is made independently in each cell.
4. Once made, the choice is stable and clonally inherited: all descendants of a cell keep the same X inactive.

A female mammal is therefore a mosaic of two cell populations, one expressing her maternal X and one expressing her paternal X. The rule extends to people with extra X chromosomes: cells keep exactly one X active and inactivate all the others, so the number of Barr bodies equals the number of X chromosomes minus one (the n minus 1 rule).

How an X is silenced

X inactivation is controlled by a region of the X called the X inactivation center (XIC). Its key gene is XIST (X inactive specific transcript), which encodes a long noncoding RNA of about 17,000 nucleotides that is never translated. The process can be pictured in steps:

1. Counting. The cell senses how many X chromosomes it has relative to its autosome sets. If there is more than one X, inactivation proceeds.
2. Choice. One X is chosen to stay active. On the future inactive X, XIST is switched on; on the future active X, XIST is kept off. In mice, an antisense transcript called Tsix, made from the opposite DNA strand across Xist, helps repress Xist on the active X.
3. Spreading. XIST RNA coats the chromosome that makes it, working only in cis (on its own chromosome). It spreads outward from the XIC, using repetitive sequences such as LINE elements as way stations.
4. Silencing and maintenance. XIST recruits proteins that remove active histone marks and add repressive ones, especially trimethylation of histone H3 at lysine 27 by Polycomb repressive complex 2. The inactive X gains the histone variant macroH2A, its CpG island promoters become methylated, and it replicates late in S phase. DNA methylation makes the silent state heritable through cell division.

In the germ line the process is reversed. In the developing oocyte, the inactive X is reactivated so that each egg carries a working X. In mouse extraembryonic tissues and in all tissues of marsupials, inactivation is imprinted: the paternal X is always the one silenced.

Escape from inactivation

Inactivation is not complete. In humans about 15 percent of X linked genes escape inactivation and are expressed from both X chromosomes, and another 10 percent or so escape in some individuals or tissues. The escapees include genes in the pseudoautosomal regions, which have partners on the Y and so are present in two active copies in both sexes. Escape explains a puzzle: if one X is inactivated anyway, why are 45,X and 47,XXY not harmless? The answer is that people with Turner syndrome have only one copy of the escape genes instead of two, and people with Klinefelter syndrome have an extra copy. Loss of one copy of the pseudoautosomal gene SHOX, for example, accounts for much of the short stature in Turner syndrome.

Visible mosaics

The most famous mosaic is the calico or tortoiseshell cat. An X linked gene, Orange, has an allele X^O that makes orange fur and an allele X^o that allows black fur. A heterozygous female (X^OX^o) has patches of orange where the X^o chromosome was inactivated and patches of black where X^O was inactivated. White spotting from an unrelated autosomal gene adds the white areas of a calico. Because the patches depend on two different X chromosomes, calico cats are nearly always female. The rare male calico is usually XXY, and like an XXY man, he is sterile.

Human females show the same mosaicism, though usually less visibly. Women heterozygous for X linked anhidrotic ectodermal dysplasia have patches of skin without sweat glands, mapped by spraying the skin with a starch iodine indicator. Women heterozygous for G6PD deficiency have two populations of red cells, one normal and one deficient. Because inactivation is random, the proportion of cells using each X varies from woman to woman. When most cells happen to inactivate the normal X, called skewed X inactivation, a carrier can show symptoms. Such women are called manifesting heterozygotes; for example, some carriers of Duchenne muscular dystrophy develop muscle weakness or heart muscle disease.

Section 4Sex influenced and sex limited traits

Not every trait that differs between males and females is caused by a gene on a sex chromosome. Many autosomal genes are expressed differently in the two sexes because males and females have different hormonal environments. Two categories are worth distinguishing, because they produce patterns that can be mistaken for sex linkage.

Sex influenced traits

In a sex influenced trait, both sexes can express the trait, but the heterozygote's phenotype depends on its sex: the allele behaves as dominant in one sex and recessive in the other. Homozygotes have the same phenotype in both sexes.

The standard example is horns in certain sheep breeds. Let H be the allele for horns and h the allele for no horns. HH sheep of both sexes are horned and hh sheep of both sexes are hornless. Hh rams are horned, while Hh ewes are hornless. In effect H is dominant in males and recessive in females, a difference driven by testosterone. Beards in goats follow the same logic. Pattern baldness is often taught the same way, dominant in men and recessive in women. In reality many genes contribute, including variants near the X linked androgen receptor gene, so treat the one gene model as a practice simplification.

Sex limited traits

In a sex limited trait, the phenotype is expressed in only one sex, although both sexes carry and transmit the genes. Milk yield in cattle is the classic example: bulls carry genes that influence milk production and pass them to their daughters, but bulls themselves give no milk. Breeders judge a bull by his daughters' yields. Other examples include genes affecting egg production in hens, the timing of puberty in boys with familial male limited precocious puberty (an autosomal dominant activating mutation in the luteinizing hormone receptor that has no effect in carrier females), and the cock feathering trait of chickens.

In chickens, males usually have long, curved, pointed tail and neck feathers (cock feathering) and females have short, rounded feathers (hen feathering). An autosomal allele H produces hen feathering in both sexes. Females are hen feathered no matter what their genotype is, so the trait of cock feathering is limited to hh males.

Telling them apart from sex linkage

Sex influenced traits can mimic X linkage in a single generation. Cross a hornless ram (hh) to a horned ewe (HH): every lamb is Hh, so sons are horned like their mother and daughters hornless like their father, which looks like crisscross inheritance. Two tests reveal the truth. An autosomal gene can pass from father to son, which an X linked gene cannot, and the F2 of a sex influenced gene shows 3:1 in one sex and 1:3 in the other.

Section 5Mitochondrial and chloroplast inheritance, maternal effect and genomic imprinting

This section covers situations in which the two parents do not contribute equally. In extranuclear inheritance, genes in mitochondria or chloroplasts come mostly from one parent. In maternal effect, the mother's nuclear genotype, not the offspring's, decides the offspring's phenotype. In genomic imprinting, a gene inherited from one parent is silenced. They are easily confused, so learn to tell them apart.

The mitochondrial genome

Mitochondria descend from bacteria that were engulfed by an ancestral cell more than a billion and a half years ago, and they still carry a small genome of their own. The human mitochondrial DNA (mtDNA) is a circular molecule of 16,569 base pairs containing 37 genes: 13 encode polypeptides of the oxidative phosphorylation system (subunits of complexes I, III, IV and ATP synthase, but none of complex II), 22 encode transfer RNAs and 2 encode ribosomal RNAs. The genome is compact, with almost no noncoding DNA except the D loop control region, and it uses a slightly different genetic code (for example, UGA codes for tryptophan instead of stop). The other 1,000 or more proteins that a mitochondrion needs are encoded in the nucleus, made in the cytosol and imported. For that reason, many mitochondrial diseases are inherited in ordinary Mendelian patterns.

A cell holds hundreds to thousands of copies of mtDNA. Its mutation rate is much higher than that of nuclear DNA, and with no recombination this makes mtDNA useful for tracing maternal ancestry.

Maternal inheritance, heteroplasmy and the threshold

In humans and most animals, mtDNA is inherited from the mother. An egg contains roughly 100,000 or more copies of mtDNA, while the few mitochondria in the sperm midpiece are tagged with ubiquitin and destroyed in the early embryo. A mother with an mtDNA mutation therefore can pass it to all of her children, sons and daughters alike, but an affected father passes it to none.

When all mtDNA copies in a cell are identical, the cell is homoplasmic; when a cell has a mixture of normal and mutant copies, it is heteroplasmic. Three ideas explain why mitochondrial diseases are so variable, even among siblings:

• The bottleneck. During oogenesis, only a small number of mtDNA molecules from each primordial germ cell repopulate the mature egg. Random sampling means a heteroplasmic mother can produce eggs with very different proportions of mutant mtDNA, so one child may be severely affected and another barely at all.
• Replicative segregation. When a cell divides, its mitochondria are distributed to daughter cells at random, so the proportion of mutant mtDNA drifts between cell lineages and tissues over a lifetime.
• The threshold effect. A tissue shows dysfunction only when the fraction of mutant mtDNA exceeds a threshold, often 60 to 90 percent depending on the mutation. Tissues with high energy demand, such as brain, retina, skeletal muscle and heart, reach their limits first.

Chloroplast inheritance

Chloroplasts also carry their own DNA, typically a circle of 120,000 to 160,000 base pairs with roughly 100 to 130 genes for photosynthesis proteins, ribosomal RNAs and transfer RNAs. In 1909 Carl Correns studied the four o'clock plant Mirabilis jalapa, whose branches can be green, white (lacking chlorophyll) or variegated. He found that the phenotype of seedlings depended only on the branch that bore the seed, the female parent, and not at all on the pollen source. Seeds from green branches gave green seedlings, seeds from white branches gave white seedlings (which die once their seed reserves run out), and seeds from variegated branches gave a mix of green, white and variegated seedlings. The variegated branches carried a mixture of normal and defective plastids, and the egg cells received different mixtures, the plant equivalent of heteroplasmy.

Maternal effect

A maternal effect is not the same as maternal inheritance. In a maternal effect, the gene is an ordinary nuclear gene that follows Mendel's laws, but the offspring's phenotype is decided by the mother's genotype, because the mother loads the egg with mRNA and proteins made from her own genes before fertilization. Those products direct the earliest stages of development, before the embryo's own genes are switched on.

The classic example is the direction of shell coiling in the pond snail Lymnaea peregra. The allele D for right handed (dextral) coiling is dominant to d for left handed (sinistral) coiling, but the direction is set by the orientation of the spindle in the first cleavage divisions, which depends on products the mother deposited in the egg. Snails are hermaphrodites, so a single snail can be crossed or self fertilized. The key results are:

• A dd female crossed to a DD male gives F1 offspring that are all Dd, yet all are sinistral, because their mother is dd.
• Selfing the F1 gives F2 offspring in a ratio of 1 DD : 2 Dd : 1 dd, but all are dextral, because their mother (an F1 snail) is Dd.
• Selfing each F2 snail reveals the 3:1 ratio one generation late: the offspring of DD and Dd F2 snails are dextral and the offspring of dd F2 snails are sinistral. So 3/4 of F2 snails produce dextral broods and 1/4 produce sinistral broods.

Maternal effect genes are central to early development. In Drosophila, the mother deposits bicoid mRNA at the front end of the egg. Its protein gradient specifies head and thorax, and a homozygous mutant mother lays eggs that develop without heads even when the embryo carries a normal paternal allele.

Genomic imprinting

For most genes, the copy from the mother and the copy from the father are expressed equally. For a few hundred mammalian genes (estimates for humans run from about 100 to more than 200), expression depends on the parent of origin: one copy is silenced according to which parent it came from. This is genomic imprinting. An imprinted gene is "maternally imprinted" if the maternal copy is silenced (so only the paternal copy is expressed) and "paternally imprinted" if the paternal copy is silenced. Because these terms are easy to flip, "paternally expressed" or "maternally expressed" is clearer.

Imprints are set by DNA methylation at imprinting control regions (ICRs) during gamete formation. In the primordial germ cells of the embryo, old imprints are erased, and new ones are written according to the sex of the individual: a male writes paternal imprints on every chromosome he will pass on, and a female writes maternal imprints. After fertilization, imprinted methylation is protected from the wave of demethylation that resets most of the genome, and it is maintained in somatic cells for life.

The Igf2 and H19 genes on mouse chromosome 7 (human 11p15) show how an imprint works. Igf2 encodes insulin like growth factor 2, a fetal growth factor, and is expressed only from the paternal chromosome. H19, a noncoding RNA gene, is expressed only from the maternal chromosome. Between them lies an ICR. On the maternal chromosome the ICR is unmethylated and binds the insulator protein CTCF, which blocks shared downstream enhancers from reaching Igf2, so the enhancers activate H19 instead. On the paternal chromosome the ICR is methylated, CTCF cannot bind, the enhancers reach Igf2, and methylation spreading into the H19 promoter silences it. A mouse that inherits a disrupted Igf2 gene from its father is small, but one that inherits the same disrupted gene from its mother is normal, because the maternal copy was silent anyway.

Prader Willi and Angelman syndromes

The clearest human example of imprinting is a cluster of genes on chromosome 15q11 to q13. Some genes in the region, including the small nucleolar RNA cluster SNORD116, are expressed only from the paternal chromosome; one gene, UBE3A, is expressed only from the maternal chromosome in neurons. The same deletion of about 5 million base pairs therefore causes two different disorders depending on its parent of origin:

• Prader Willi syndrome results from loss of the paternal contribution: poor muscle tone and feeding problems in infancy, followed by insatiable appetite and obesity, short stature, small hands and feet, hypogonadism and mild to moderate intellectual disability. About 70 percent of cases are paternal deletions and most of the rest are maternal uniparental disomy, in which the child received two copies of chromosome 15 from the mother and none from the father.
• Angelman syndrome results from loss of the maternal contribution: severe intellectual disability, absent speech, seizures, a jerky gait and a frequently happy demeanor with easily provoked laughter. About 70 percent are maternal deletions; others arise from paternal uniparental disomy, imprinting defects or point mutations in the maternal UBE3A.

Imprinting defects at 11p15 cause Beckwith Wiedemann syndrome (overgrowth and embryonal tumor risk, often from excess IGF2) and its mirror image, Silver Russell syndrome (growth restriction). The parental conflict hypothesis proposes that imprinting evolved as a tug of war over fetal growth: paternally expressed genes such as Igf2 tend to promote it and maternally expressed genes tend to restrain it.

Words worth knowing