HSP90AA1 variants may contribute to autosomal dominant human male infertility

This study suggests that variants in the HSP90AA1 gene may act as a rare autosomal dominant cause of human male infertility, a finding supported by the identification of heterozygous pathogenic variants in infertile men despite the gene's autosomal recessive inheritance pattern in mouse models.

The Gist

The Big Picture: A Broken Factory Manager

Imagine your body is a massive, bustling factory. One of its most important jobs is making sperm. To do this, the factory needs a team of highly skilled workers and a very specific manager to keep everything running smoothly.

The gene HSP90AA1 is the blueprint for that manager. In the world of biology, this manager is a "chaperone" protein. Its job is to help other proteins fold into the right shape so they can do their work. Without this manager, the factory floor gets chaotic, and the sperm production line grinds to a halt.

This study asked a simple question: If the blueprint for this manager is damaged, does it cause infertility in human men?

The Mystery: Mice vs. Humans

The researchers started with a clue from mice.

• The Mouse Story: In mice, if you completely destroy the blueprint for this manager (knock it out), the male mice become sterile. However, if you only damage one copy of the blueprint (leaving the other one working), the mice are perfectly fine. This suggested the problem is recessive (you need two broken copies to get sick).
• The Human Puzzle: But humans are tricky. When the researchers looked at computer models of the human gene, the data screamed something different. The human version of this gene is so sensitive that having just one broken copy might be enough to cause trouble. This is called a dominant inheritance pattern.

So, the team had to investigate: Is this gene like a mouse (needs two broken copies) or a human (one broken copy is enough)?

The Investigation: Searching the Library

The team looked at the genetic "libraries" (DNA data) of over 2,300 men who were struggling to have children. They were hunting for typos (variants) in the HSP90AA1 blueprint.

1. The "Homozygous" Hunt (Two Broken Copies)
They found one man with a specific typo where he had two broken copies of the gene.

• The Test: To see if this typo was the real culprit, they built a mouse with the exact same typo.
• The Result: The mouse was perfectly healthy and had plenty of babies.
• The Conclusion: This specific typo was a "false alarm." It wasn't the cause of the man's infertility.

2. The "Heterozygous" Hunt (One Broken Copy)
Since the mouse data suggested the human gene might be sensitive to just one broken copy, they looked for men who had only one typo.

• The Findings: They found five men with different types of damage to just one copy of the gene.
  • One man had a "frameshift" error (like a sentence where a letter is deleted, scrambling the rest of the message).
  • Four men had "missense" errors (like a typo changing a word to the wrong one, e.g., changing "run" to "fun").
• The Symptoms: All these men had severe sperm problems, ranging from having zero sperm (azoospermia) to having very few (cryptozoospermia).
• The Theory: Even though they had one good copy of the gene, the "broken" copy was likely causing enough trouble to disrupt the factory. It's like having a manager who is half-asleep; the factory still runs, but it's inefficient and eventually stops producing.

The "Why" and The "How"

Why does this matter?

• Most infertility genes are recessive: Usually, you need to inherit a broken gene from both parents to be affected.
• This gene might be dominant: This study suggests HSP90AA1 might be one of the rare genes where inheriting a broken copy from just one parent is enough to cause infertility. This is a big deal because it changes how doctors might test for these issues in the future.

The Catch (Limitations)

The researchers are careful not to jump to conclusions yet.

• The Mouse Model Didn't Match: The mouse with the "broken" gene was fine. This makes it hard to prove 100% that the human gene works exactly the same way.
• Need More Proof: They need to find more families with this specific gene issue to confirm that it really is the cause. Right now, it's a very strong suspect, but not a confirmed criminal.

The Takeaway

Think of this study as a detective finding a new suspect in a cold case.

• The Suspect: The HSP90AA1 gene.
• The Crime: Male infertility.
• The Clue: Men with just one damaged copy of this gene seem to have trouble making sperm.
• The Next Step: The detectives need to find more witnesses (more patients) to confirm the suspect is guilty before they can arrest them (make it a standard medical test).

If this is confirmed, it opens a new door for diagnosing infertility, helping couples understand why they can't conceive and potentially guiding them toward better treatments or family planning options.

Technical Summary

1. Problem Statement

Male infertility is a heterogeneous condition affecting ~15% of couples, with a male factor present in roughly half of these cases. While over 300 genes have been linked to male infertility, the majority follow an autosomal recessive mode of inheritance (MOI). A significant diagnostic gap remains for men with non-obstructive azoospermia (NOA) or severe oligozoospermia.

• The Candidate: HSP90AA1 encodes the heat shock protein 90 alpha (HSP90α), a molecular chaperone essential for spermatogenesis.
• The Discrepancy: In mice, biallelic knockout of Hsp90aa1 causes male-specific azoospermia (recessive), while heterozygous mice are fertile. However, human genetic constraint metrics suggest HSP90AA1 might be intolerant to haploinsufficiency, hinting at a potential autosomal dominant MOI in humans, similar to the known gene DMRT1.
• Objective: To investigate whether variants in HSP90AA1 cause human male infertility and to determine the mode of inheritance.

2. Methodology

The study employed a multi-faceted approach combining human genomics, bioinformatics, and mouse modeling.

• Human Cohort Screening:

  • Dataset: Exome/genome sequencing data from the MERGE cohort (>2,300 infertile men), primarily consisting of azoospermic, cryptozoospermic, and severe oligozoospermic patients.
  • Filtering Strategy: Variants were filtered for:
    • Minor Allele Frequency (MAF) ≤ 0.001 (gnomAD v2.1.1).
    • CADD score ≥ 20 for missense variants.
    • Exclusion of patients with known genetic causes of infertility.
    • Screening for both heterozygous and homozygous/compound heterozygous variants.
  • In Silico Prediction: Pathogenicity was assessed using AlphaMissense, DOMINO (for MOI prediction), and constraint metrics (pLI, LOEUF, pHaplo).

• Mouse Model Generation:

  • Target: The specific homozygous missense variant found in a human patient: c.605G>A p.(Arg202Lys).
  • Technique: CRISPR-Cas9 gene editing in B6CBAF1/Crl zygotes to create the Hsp90aa1^R202K allele.
  • Phenotyping:
    • Fertility: Plug-mating assays to count litter sizes.
    • Histology: Testicular and epididymal sections stained with Periodic Acid-Schiff (PAS) to assess spermatogenesis stages.
    • Immunofluorescence (IF): Staining for HSP90AA1 protein expression and meiotic markers (SCP1, SCP3) to check for synaptonemal complex defects.

• Human Histology:

  • Testicular biopsies from identified patients underwent PAS staining to characterize the spermatogenic phenotype (e.g., Sertoli Cell Only, Hypospermatogenesis).

3. Key Results

A. Mode of Inheritance Analysis

• Mouse vs. Human: Mouse data supports a recessive MOI (heterozygotes are fertile). However, human constraint metrics strongly suggest a dominant MOI:
  • pLI = 1 (indicating haploinsufficiency).
  • LOEUF = 0.24 (highly intolerant to loss-of-function).
  • pHaplo = 0.96 and DOMINO prediction: "Very likely dominant."
• Conclusion: The study screened for both heterozygous and homozygous variants in humans.

B. The Homozygous Variant (c.605G>A p.Arg202Lys)

• Patient: Identified in an azoospermic man (M3272) with a Sertoli Cell Only (SCO) phenotype and consanguineous parents.
• Mouse Validation: Homozygous Hsp90aa1^R202K/R202K mice were generated.
  • Outcome: Mice were fertile with normal litter sizes, normal testicular weights, and normal histology (complete spermatogenesis).
  • Protein/Meiosis: No differences in HSP90AA1 expression or synaptonemal complex assembly were observed.
• Conclusion: The p.(Arg202Lys) variant is likely benign and not the cause of the patient's infertility.

C. Heterozygous Variants (The Primary Finding)

The study identified five heterozygous variants in HSP90AA1 in men with severe spermatogenic failure:

1. c.1068_1071del p.(Lys357ArgfsTer20): A frameshift variant in Patient M1551 (Azoospermia, Hypospermatogenesis). Predicted to cause nonsense-mediated decay (LoF).
2. c.133C>G p.(Leu45Val): Missense in the N-terminal ATPase domain (Patient M3510, Azoospermia).
3. c.1445C>T p.(Thr482Ile): Missense in the middle domain (Patient M2503, Cryptozoospermia).
4. c.1503G>C p.(Gln501His): Missense in the middle domain (Patient M2011, Azoospermia).
5. c.2150C>T p.(Pro717Leu): Missense in the C-terminal dimerization domain (Patient M2643, Cryptozoospermia).

• Phenotypic Correlation: All patients exhibited severe spermatogenic defects (azoospermia or cryptozoospermia) with histology showing hypospermatogenesis (arrest at spermatocyte stage with rare post-meiotic cells).
• Prediction: AlphaMissense predicted most missense variants as "Likely Pathogenic" or "Ambiguous," except for p.(Gln501His).

4. Key Contributions

1. Novel Candidate Gene: Establishes HSP90AA1 as a novel candidate gene for human male infertility.
2. Dominant MOI Hypothesis: Provides evidence that HSP90AA1 may follow an autosomal dominant mode of inheritance in humans, contrasting with the recessive pattern in mice. This highlights the importance of cross-species validation for inheritance patterns.
3. Variant Exclusion: Successfully ruled out a specific homozygous missense variant (p.Arg202Lys) via mouse modeling, demonstrating the necessity of functional validation.
4. Clinical Phenotype: Links heterozygous HSP90AA1 variants to a specific phenotype of hypospermatogenesis/arrest, suggesting that haploinsufficiency reduces but does not completely abolish spermatogenic capacity.

5. Significance and Limitations

• Significance:
  • Expands the genetic landscape of male infertility beyond the typical recessive genes.
  • Suggests that HSP90AA1 haploinsufficiency disrupts the chaperoning of the Androgen Receptor (AR) in spermatogonia, leading to meiotic arrest.
  • Offers a potential diagnostic target for men with unexplained azoospermia/cryptozoospermia.
• Limitations:
  • Evidence Level: Currently classified as "limited evidence" (ClinGen) because functional studies for the specific human missense variants were not performed, and segregation analysis in families was not possible for all cases.
  • Replication: Findings need replication in independent cohorts to confirm the dominant MOI.
  • Mouse Model: The mouse model for the specific human missense variants (other than R202K) has not yet been generated to confirm pathogenicity.

Conclusion: The study proposes HSP90AA1 as a rare autosomal dominant cause of human male infertility, driven by heterozygous loss-of-function or missense variants that impair spermatogenesis, while emphasizing the need for further validation to move from candidate status to clinical diagnostic utility.