This article appears in The Journal of Clinical Endocrinology & Metabolism, published January 5, 2011, 10.1210/jc.2010-2292

ContextPrimary ovarian insufficiency (POI) results from a premature loss of oocytes, causing infertility and early menopause. The etiology of POI remains unknown in a majority of cases.ObjectiveTo identify candidate genes in families affected by POI.DesignThis was a family-based genetic study.SettingThe study was performed at two academic institutions.Patients and Other ParticipantsA family with four generations of women affected by POI (n = 5). Four of these women, three with an associated autoimmune diagnosis, were studied. The controls (n = 387) were recruited for health in old age.InterventionWhole-genome sequencing was performed.Main Outcome MeasureCandidate genes were identified by comparing gene mutations in three family members and 387 control subjects analyzed simultaneously using the pedigree Variant Annotation, Analysis and Search Tool. Data were also compared with that in publicly available databases.ResultsWe identified a heterozygous nonsense mutation in a subunit of RNA polymerase II (POLR2C) that synthesizes messenger RNA. A rare sequence variant in POLR2C was also identified in one of 96 women with sporadic POI. POLR2C expression was decreased in the proband compared with women with POI from another cause. Knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation.ConclusionsThese data support a role for RNA polymerase II mutations as candidates in the etiology of POI. The current data also support results from genome-wide association studies that hypothesize a role for RNA polymerase II subunits in age at menopause in the population.

Context Functional hypothalamic amenorrhea (HA) is a common, acquired form of hypogonadotropic hypogonadism that occurs in the setting of energy deficits and/or stress. Variability in individual susceptibility to these stressors, HA heritability, and previous identification of several rare sequence variants (RSVs) in genes associated with the rare disorder, isolated hypogonadotropic hypogonadism (IHH), in individuals with HA suggest a possible genetic contribution to HA susceptibility. Objective We sought to determine whether the burden of RSVs in IHH-related genes is greater in women with HA than controls. Design We compared patients with HA to control women. Setting The study was conducted at secondary referral centers. Patients and Other Participants Women with HA (n=106) and control women (ClinSeq study; n=468). Interventions We performed exome sequencing in all patients and controls. Main Outcome Measure(s) The frequency of RSVs in 53 IHH-associated genes was determined using rare variant burden and association tests. Results RSVs were overrepresented in women with HA compared with controls (P = 0.007). Seventy-eight heterozygous RSVs in 33 genes were identified in 58 women with HA (36.8% of alleles) compared to 255 RSVs in 41 genes among 200 control women (27.2%). Conclusions Women with HA are enriched for RSVs in genes that cause IHH suggesting that variation in genes associated with GnRH neuronal ontogeny and function may be a major determinant of individual susceptibility to developing HA in the face of diet, exercise and/or stress.

CONTEXT Isolated prolactin deficiency is a rare disorder manifesting as absence of puerperal lactation. We identified a two-generation family with 3 women experiencing alactogenesis. OBJECTIVE We hypothesized a heterozygous genetic mutation. DESIGN This was a family-based study. PATIENTS Two generations of women (proband, sister and niece) with puerperal alactogenesis and one control were studied. Prolactin levels in the three women ranged from 0.618 to 1.4 ng/mL (2.8-29.2 ng/mL). All the women had regular menstrual cycles during their reproductive years. The niece required fertility treatment to become pregnant and the proband and sister underwent menopause before age 45 years. INTERVENTION PRL exons 1-5 were sequenced. MAIN OUTCOME MEASURE We sought a heterozygous, deleterious gene variant with functional consequences. RESULTS We identified a heterozygous mutation (c.658C>T) changing CGA to TGA (p.Arg220Ter) in exon 5 of the prolactin gene. Transfection of PRL containing the stop gain mutation resulted in similar intracellular prolactin levels compared to PRL wild type, but little detectable immunoactive or bioactive prolactin in conditioned medium. Prolactin secretion was also impaired by a PRL stop gain mutation deleting both of the terminal cysteine amino acids (c.652A>T; p.Lys218Ter). CONCLUSIONS This is the first report of a PRL mutation causing familial prolactin deficiency and alactogenesis. The loss of the terminal cysteine resulted in failure of prolactin secretion. Secretion was not rescued by deleting the penultimate cysteine, with which it forms a disulfide bond. These data suggest that the PRL C terminal is critical for protein secretion.

BACKGROUND: Isolated prolactin deficiency is a rare disorder manifesting as absence of puerperal lactation. We identified a family with multiple members who had alactogenesis and examined genetic causes. SUBJECTS: Three women in one family (proband, sister and niece) reported puerperal alactogenesis, i.e. no milk production postpartum. All the women had regular menstrual cycles during their reproductive years. The proband, currently 66 yrs, had 7 pregnancies and 2 live births. Prolactin level was 1.16 ng/mL (2.8-29.2 ng/mL) at the age of 47 yrs. The sister, currently 73 yrs, had 3 children. Her prolactin level was 1.4 ng/mL (2.8-29.2 ng/mL) at the age of 64 yrs. Both women had menopause before age 45 years. The niece, currently 43 years old, had infertility of unknown cause and conceived 2 children with fertility treatment. She had prolactin levels of 1.13 ng/mL, 0.618 ng/mL and 0.759 ng/mL at the age of 34-36 yrs. METHODS: DNA and mRNA were extracted from blood of all three women and a fertile control with a history of normal lactation. PRL exons 1-6 were Sanger sequenced from the DNA. cDNA was subjected to RT-PCR with primers spanning intron 5 and the terminal portion of exon 6. RT-PCR was analyzed using the 2 -ΔΔC T method. Prolactin mRNA expression was compared among the affected women and the control subject. RESULTS: We identified a heterozygous mutation (c.658C>T) changing CGA (arginine) to TGA (stop codon) (p.Arg220Ter) in exon 6 of the prolactin gene. PRL mRNA expression (0.011±0.003 vs. 1.0±0.0; p<0.001) and terminal exon 6 expression (0.0027±0.0028 vs. 0.99±0.05; p<0.001) was decreased compared to control mRNA expression. DISCUSSION: We have identified a genetic cause of puerperal alactogenesis resulting from a stop-gain mutation in the prolactin gene ( PRL ). PRL mRNA expression is decreased in these subjects. These findings suggest that the mutated PRL may undergo nonsense decay. The low circulating prolactin resulted in inability to breastfeed. It may also be the cause of infertility and early menopause, but further studies are needed. CONCLUSION: We report a stop-gain mutation in exon 6 of the prolactin gene ( PRL ) resulting in familial puerperal alactogenesis.