Study populations and DNA samples are as previously described 28. Briefly, DNA samples from the premature ovarian failure group were collected from women with premature ovarian failure (age range: 13–41 years). Age at menopause was defined as amenorrhea for at least four months, with two serum FSH (follicle stimulating hormone) levels in the menopausal range one month apart 29. Causes of ovarian failure such as X chromosome translocations and deletions were excluded. DNA samples from the ovarian failure/menopause group were collected from women with ovarian failure/early menopause prior to or after the age of 46, as described 2. One third of the women in this population reported an early menopause and a family history of early menopause (prior to age 46, N = 108), one third reported an early menopause and no family history (N = 108), and the remaining third reported menopause after the age of 46 or were still menstruating at age 46, and served as controls (N = 108). Samples were obtained only from the women surveyed and not from their family members. DNA samples from the infertile male group were collected from infertile men; each man in this group had a complete history, physical exam, karyotype, semen analysis performed according to WHO criteria, and testicular biopsy when appropriate 30. In the case of multiple semen analyses with different reported values, the semen sample with the highest sperm count was included in statistical models.

DNA extraction and genotype were performed as described 28. The entire coding region of DAZL was sequenced, including all exons and flanking regions. Primers, primer concentrations, and PCR conditions were as indicated 28. PCR primers were designed using Primer3, with or without the human repeat mispriming library 31. Sequencher v4.1.4 (Gene Codes, Ann Arbor, MI), Seqman (DNAStar Inc., Madison, WI) and Mutation Surveyor v2.1 (SoftGenetics, State College, PA) were used to align sequences and to identify polymorphic bases.

This mutation changes a highly conserved proline to a histidine in the N-terminus of the protein, just upstream of the highly-conserved RNA Recognition Motif (RRM). Although proline is usually considered a hydrophobic amino acid whose unique cyclic structure causes it to influence protein architecture, its secondary amino (NH₂) group predisposes this residue to being on the surface of the protein exposed to water. Histidine is also a bulky amino acid that can be either neutrally or positively charged, depending on its environment. Overall, since both amino acids are large and generally localized to the surface of the protein, this amino acid change is most likely to affect the structure of the protein surface. In this study, we identified one woman who was heterozygous for this mutation, with onset of menopause at age 45, and no family history of early menopause. She was fertile, having had three children prior to menopause.

This amino acid change was identified in two patients: a male homozygous for the mutation who presented with azoospermia and a female heterozygous for the mutation who reported an early menopause at age 44. Notably, asparagine is a negatively charged hydrophilic residue, while cysteine contains the hydrophobic and highly reactive sulfhydryl group. This particular change lies in exon 2 just outside of the RNA-binding domain; the substitution of this hydrophilic residue for a hydrophobic residue may impact protein folding. Interestingly, in this case, the mutation may act in a dose-dependent manner: the woman with one copy of the mutation had a less severe phenotype than the male with two copies of the mutation.

This isoleucine is part of the RNA-binding domain (the RRM or RNA Recognition Motif). It is five amino acids upstream of the RNP2 sequence which is one of the most highly conserved parts of the DAZL protein. Isoleucine and alanine are both classified as hydrophobic amino acids; however, alanine, with its shorter side chain, is not as hydrophobic as isoleucine. The structure of the homologous RNA binding domain in another protein has been deduced, and in that structure, the amino acid corresponding to the isoleucine forms a hydrogen bond with another amino acid in the structure 32. The shorter side chain of alanine would be expected to disrupt this hydrogen bond. This mutation was only identified in a woman who experienced a spontaneous early age at onset of menopause at age 43. She was heterozygous for the mutation and had one child.

Arg115 is near the carboxy-terminal end of the RRM in a region that interacts with several other proteins 33. This mutation results in the substitution of a basic, hydrophilic amino acid for the simplest amino acid, glycine. By sequence comparison to the RRM of another RNA-binding protein, the HuD protein which has been crystallized bound to RNA, it appears that this amino acid may be key to making side-chain and main-chain connections to the RNA and the other RRM in this protein 34. As DAZL is also known to homodimerize, this amino acid may be important for both RNA binding and protein-protein interactions. Substitution of arginine by glycine should severely disrupt normal interactions with this amino acid. Accordingly, the one patient we identified with this mutation was homozygous for this change, and experienced premature ovarian failure at age 34, having borne no children.

Our screen of the DAZL gene for sequence variations that are associated with male and female fertility produced not only a number of interesting common SNPs or variants 28, but also intriguing rare missense mutations, as reported here. None of these mutations were found in the 108 control women that had a normal menopause or were still menstruating at age 46, suggesting they may be associated with impaired germ cell development and fertility. Nonetheless, additional data is required; for example, it would be most useful to independently screen a second group of male and female control chromosomes to determine whether these mutations are found in individuals with "normal" fertility/reproductive characteristics. This is especially merited given that these mutations were clustered in exons that form part of the RRM. While it is possible that this region of the protein is simply highly variable, it is more likely that our population is enriched for mutations in a functionally-important part of the protein. In other RRM-containing proteins, many of the residues surrounding the RRM motif are also involved in RNA binding specificity and thus, even those mutations that are adjacent to the RRM may alter proper protein function 35.

Interestingly, we observed that possessing these putative missense mutations, especially a heterozygous missense mutation, was compatible with producing offspring. Each patient that was heterozygous for a missense mutation was able to produce at least one child. In contrast, the patient that was homozygous for the Arg115Gly mutation experienced 46,XX spontaneous premature ovarian failure at the early age of 34 years and did not bear any children; similarly, the male patient that was homozygous for the Asn10Cys mutation produced no sperm. This is consistent with data from the mouse, where mice heterozygous for a Dazl knockout allele are fertile (though it is currently debated whether they may be subfertile) and homozygous mutant mice are infertile in both sexes 17.

Recently, a number of potential RNA targets for DAZL have been identified, making it possible to test the effects of mutations on RNA-binding in vitro, in the future 36373839. In addition, as this region is known to be involved in DAZL homodimerization and binding to other proteins, it would be of interest to determine whether these mutations, like Arg115Gly for example, affect the ability of DAZL to interact with other protein partners 334041.