Genes and Genetic Disorders of the Y Chromosome

Isolation of the Testis Determining Factor (TDF)

• In the 1950s, karyotyping indicated that the Y chromosome contained a locus that triggers the development of the undifferentiated gonad into testes.

• This locus was initially termed the testis determining factor (TDF).

• In the 1980s and 90s, the responsible gene was isolated.

• Genes were initially mapped by linkage, observing the co-segregation of traits with markers in families.

• The frequency of recombination between the phenotype and the markers was used to identify the genetic interval.

• Once an interval was identified, cloning and sequencing were used to identify suitable candidate genes. This positional cloning approach was very time-consuming and costly.

• Due to the absence of recombination on the male-specific region of the Y chromosome, the positional cloning approach could not be used.

• Deletion mapping was used instead, leveraging karyotyping data showing individuals with aberrant Y chromosomes and sex-reversed individuals (XX males and XY females).

Deletion Mapping Approach

• Malcolm Ferguson Smith hypothesized that the testis factor had been transferred to the X chromosome by an aberrant recombination event in XX males.

• The reciprocal event would result in XY females.

• About 40% of breakpoints from these events fall within a small region of high XY homology, approximately 7 megabases proximal to the Par1 boundary on the short arm of the sex chromosomes.

• The portion of the Y chromosome that had been translocated was examined to determine the male phenotype.

• The smallest possible interval within which the testis-determining factor must reside was identified.

• This approach led to the identification of a 35 kilobase long interval.

Identification of the SRY Gene

• The 35 kilobase region was cloned into approximately 500 base pair long fragments.

• Each fragment was used as a probe against a Southern blot of DNA from males and females from various mammalian species.

• Evolutionarily conserved fragments were identified.

• Sequence analysis of these overlapping fragments identified a gene encoding a DNA binding domain.

Functional Analysis of SRY

• The gene was expressed in the correct location and time in mice.

• De novo mutations in the gene were observed in XY females.

• Around 80% of XX males were found to carry the SRY gene.

• Definitive proof came when the introduction of the mouse SRY gene into a chromosomally female mouse embryo resulted in the development of a normal male.

• Other genes needed for male development are located on the X chromosome and/or autosomes.

• The SRY gene is small, with a single exon, and its expression is not limited to the gonad.

• The SRY protein encodes a high mobility group (HMG) box where XY female mutations cluster.

• This HMG box, composed of three helices, binds to and bends DNA.

• SRY belongs to the SOX family of genes (SRY box related).

• The SOX family encodes regulators of development, including SOX9, also involved in sex determination.

Mechanism of Sex Determination

• Transient expression of SRY in the indifferent gonad triggers a cascade of gene interactions orchestrated by SOX9, leading to testis formation.

• SRY acts as an enhancer of SOX9 gene expression, upregulating SOX9, which is located on the long arm of chromosome 17.

• SOX9 supplants SRY to control its own expression and initiate a cascade of gene interactions for testis formation.
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Other Types of Sex Reversal

• Sex reversal can occur due to X-linked or autosomal genes involved in sex determination.

• Around 75% of XY females have an intact functional SRY gene but possess loss-of-function mutations in genes downstream of SRY.

• 20% of XX males do not have SRY but possess gain-of-function mutations in genes downstream.

• Females with complete androgen insensitivity syndrome due to mutations in the androgen receptor gene on the X chromosome cannot respond to androgen.

• Congenital adrenal hyperplasia, due to mutations on an autosomal gene, results in overproduction of androgens, leading to a male phenotype in XX individuals.

Diseases of the Y Chromosome

• Hairy ears were initially believed to be a Y-linked trait.

• However, females can also be affected, and it is no longer considered a Y-linked trait.

• In 2004, non-syndromic deafness was reported as a Mendelian Y-linked trait but was later found to be due to a complex rearrangement involving duplications of non-contiguous portions of the Y chromosome and insertion of 160 kilobases from chromosome 1.

• The chromosome 1 segment contains five genes part of a hearing impairment locus.

• This appears to be a private mutation segregating in a single large family.

Role of the Y Chromosome in Common Complex Diseases

• Hypertension is a candidate involving both autosomes and the Y chromosome in rat models.

• SRY itself has been postulated as the Y chromosome mediator in rat models. However, this has not been replicated in human studies.

• The UK Biobank cohort, containing 230,000 men SNP typed for 800,000 Y chromosome markers, is being used to establish links with medical-related phenotypic information using a phylogenetic approach.

• A significantly higher risk for coronary artery disease has been found for men of particular lineages of the Y chromosome (e.g., Haplogroup I).

• Differences in gene expression levels have been noted in genes involved in inflammatory or immune signaling cascades.

• Haplogroup I is associated with altered immunity in the context of HIV infection in Americans of European descent.

• The specific Y chromosome genes implicated have not been established.

Y-Linked Infertility

• Azoospermia factor (AZF) was identified by karyotyping infertile men in the 1970s.

• In the 1990s, candidate was DFFRY. However, normal fertile males can have the entire gene deleted.

• Three separate genomic regions are now recognized to be involved in male fertility. These regions are large (at least 1 megabase long intervals), contain a mix of genes, and lead to different causes of infertility.

• Less than 5% of male infertility cases result from deletion of the AZFA region containing single-copy genes.

Genomic Disorders and Infertility

• AZF phenotypes are examples of genomic disorders caused by underlying structural features of the genome, such as highly similar repeats (paralogues or segmental duplications).

• These repeats may misalign, allowing non-allelic homologous recombination to occur, resulting in deletions, duplications, or inversions.

• Duplications and deletions arise when repeats are in the same direction (direct repeats).

• Inversions arise intramolecularly via inverted repeats.

• 90% of AZFA cases result from deletion of around 800 kilobases between two 10 kilobase long dispersed repeats (Herve elements).

• Many cases of AZFC infertility are associated with aberrant recombination between longer and more similar segmental duplications.

• Longer and more similar repeats are more likely to misalign and cause rearrangements.

Unusual Genomic Landscape of the Y Chromosome

• The Y chromosome has a higher proportion of segmental duplications, also known as ampliconic sequences.

• The long arm consists of five pairs of palindromic sequences.

• The arms of these palindromes are more similar to each other due to non-reciprocal exchanges (gene conversion events).

• Only half of the transcribed sequences are protein-coding and fall into nine gene families restricted in their expression to the testis (18 single-copy genes).

• A comparatively large number of pseudogenes exist due to the differentiation of the Y from the historical autosome.

• The Y chromosome shows functional specialization with a non-random collection of genes.

• The function of many untranslated transcripts has yet to be established.