Delayed puberty and estrogen resistance in a woman with estrogen receptor α variant.

Although androgen resistance has been characterized in men with a normal chromosome complement and mutations in the androgen-receptor gene, a mutation in the gene encoding estrogen receptor α (ESR1) was previously described only in one man and not, to our knowledge, in a woman. We now describe an 18-year-old woman without breast development and with markedly elevated serum levels of estrogens and bilateral multicystic ovaries. She was found to have a homozygous loss-of-function ESR1 mutation in a completely conserved residue that interferes with estrogen signaling. Her clinical presentation was similar to that in the mouse orthologue knockout. This case shows that disruption of ESR1 causes profound estrogen resistance in women. (Funded by the National Institutes of Health.).

Effect of Deep Brain Stimulation on Swallowing Function: A Systematic Review.

The effect of deep brain stimulation (DBS) on swallowing function in movement disorders is unclear. Here, we systematically reviewed this topic by searching keywords following PICOS strategy of problem (swallowing or swallow or dysphagia or aspiration) and intervention (deep brain stimulation, or DBS) in the PubMed and Web of Science in English in April 2020, with comparators [subthalamic nucleus (STN), globus pallidus interna (GPi), ventralis intermedius, (ViM), post-subthalamic area, or caudal zona incerta (PSA/cZi); ON/OFF DBS state/settings, ON/OFF medication state, Parkinson's disease (PD), dystonia, tremor], outcomes (swallowing function measures, subjective/objective) and study types (good quality original studies) in mind. We found that STN DBS at usual high-frequency stimulation could have beneficial effect (more so on subjective measures and/or OFF medication), no effect, or detrimental effect (more so on objective measures and/or ON medication) on swallowing function in patients with PD, while low-frequency stimulation (LFS) could have beneficial effect on swallowing function in patients with freezing of gait. GPi DBS could have a beneficial effect (regardless of medication state and outcome measures) or no effect, but no detrimental effect, on swallowing function in PD. GPi DBS also has beneficial effects on swallowing function in majority of the studies on Meige syndrome but not in other diseases with dystonia. PSA/cZi DBS rarely has detrimental effect on swallowing functions in patients with PD or tremor. There is limited information on ViM to assess. Information on swallowing function by DBS remains limited. Well-designed studies and direct comparison of targets are further needed.

Androgens increase gonadotropin-releasing hormone neuron firing activity in females and interfere with progesterone negative feedback.

GnRH neurons are the central regulators of fertility, and their activity is modulated by steroid feedback. In women with hyperandrogenemic infertility and in animal models of these disorders, elevated androgen levels interfere with progesterone (P) negative feedback. Our previous work showed that steroids altered the frequency and amplitude of gamma-aminobutyric acid (GABA) transmission to GnRH neurons. Specifically, P inhibited GABA transmission, which can excite GnRH neurons, whereas dihydrotestosterone (DHT) increased GABA transmission. In this study the GnRH neuron firing rate was examined in the same animal models. Adult (>2 months) female mice were ovariectomized and treated for 8-12 d with implants containing estradiol (E), E and P, E and DHT, or E, P, and DHT. Targeted extracellular recordings were used to examine the long-term firing activity of green fluorescent protein-identified GnRH neurons in brain slices from these mice. In comparing E alone to E plus P animals, P increased the percentage of time that GnRH neurons were quiescent and reduced the area under the curve of the firing rate and the instantaneous firing frequency, suggesting that P provides additional negative feedback over E alone. The addition of DHT markedly increased GnRH neuron activity in both the presence and absence of P. DHT also altered the firing pattern of GnRH neurons, such that peaks in the firing rate detected by the Cluster8 algorithm were approximately doubled in frequency and amplitude. These data support and extend our previous findings and are consistent with the hypothesis that the changes in GABAergic transmission observed in these animal models impact upon the activity of GnRH neurons, and central androgen action probably stimulates GnRH release.

Targeted next generation sequencing approach identifies eighteen new candidate genes in normosmic hypogonadotropic hypogonadism and Kallmann syndrome.

The genetic basis is unknown for ∼60% of normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS). DNAs from (17 male and 31 female) nHH/KS patients were analyzed by targeted next generation sequencing (NGS) of 261 genes involved in hypothalamic, pituitary, and/or olfactory pathways, or suggested by chromosome rearrangements. Selected variants were subjected to Sanger DNA sequencing, the gold standard. The frequency of Sanger-confirmed variants was determined using the ExAC database. Variants were classified as likely pathogenic (frameshift, nonsense, and splice site) or predicted pathogenic (nonsynonymous missense). Two novel FGFR1 mutations were identified, as were 18 new candidate genes including: AMN1, CCKBR, CRY1, CXCR4, FGF13, GAP43, GLI3, JAG1, NOS1, MASTL, NOTCH1, NRP2, PALM2, PDE3A, PLEKHA5, RD3, and TRAPPC9, and TSPAN11. Digenic and trigenic variants were found in 8/48 (16.7%) and 1/48 (2.1%) patients, respectively. NGS with confirmation by Sanger sequencing resulted in the identification of new causative FGFR1 gene mutations and suggested 18 new candidate genes in nHH/KS.

NELF knockout is associated with impaired pubertal development and subfertility.

Puberty and reproduction require proper signaling of the hypothalamic-pituitary-gonadal axis controlled by gonadotropin-releasing hormone (GnRH) neurons, which arise in the olfactory placode region and migrate along olfactory axons to the hypothalamus. Factors adversely affecting GnRH neuron specification, migration, and function lead to delayed puberty and infertility. Nasal embryonic luteinizing hormone-releasing factor (NELF) is a predominantly nuclear protein. NELF mutations have been demonstrated in patients with hypogonadotropic hypogonadism, but biallelic mutations are rare and heterozygous NELF mutations typically co-exist with mutations in another gene. Our previous studies in immortalized GnRH neurons supported a role for NELF in GnRH neuron migration. To better understand the physiology of NELF, a homozygous Nelf knockout (KO) mouse model was generated. Our findings indicate that female Nelf KO mice have delayed vaginal opening but no delay in time to first estrus, decreased uterine weight, and reduced GnRH neuron number. In contrast, male mice were normal at puberty. Both sexes of mice had impaired fertility manifested as reduced mean litter size. These data support that NELF has important reproductive functions. The milder than expected phenotype of KO mice also recapitulates the human phenotype since heterozygous NELF mutations usually require an additional mutation in a second gene to result in hypogonadotropic hypogonadism.

Differential expression of nasal embryonic LHRH factor (NELF) variants in immortalized GnRH neuronal cell lines.

NELF, a protein identified in migratory GnRH neurons, is predominantly nuclear and alternatively spliced. However, specific NELF splice variants expressed in immortalized GnRH neuronal cell lines from mouse and human are not known. RNA from migratory (GN11 and NLT) and postmigratory (GT1-7) cells in mouse, and (FNCB4-hTERT) cells in human was subjected to RT-PCR. RT-PCR products were cloned, electrophoresed on denaturing gradient gels and sequenced. In addition, quantitative RT-PCR was performed using variant-specific primers. Western blot and immunofluorescence using confocal microscopy were performed for selected variants. Nelf variant 2 (v2), which contains a nuclear localization signal (NLS), was the predominant variant in all mouse and human GnRH neurons. Variants without a NLS (v3 in mouse; v4 in human) were identified. In mouse, v2 protein expression was nuclear, while v3 was non-nuclear. In mouse GnRH neurons, six Nelf splice variant transcripts were identified, including three previously unreported variants. In human, four NELF variant transcripts were observed. In both mouse and human, nuclear and non-nuclear variant transcript and protein were identified, explaining variable NELF cellular localization.

The prevalence of digenic mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome.

OBJECTIVE: To determine the prevalence of digenic mutations in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). DESIGN: Molecular analysis of DNA in IHH/KS patients. SETTING: Academic medical center. PATIENT(S): Twenty-four IHH/KS patients with a known mutation (group 1) and 24 IHH/KS patients with no known mutation (group 2). INTERVENTION(S): DNA from IHH/KS patients was subjected to polymerase chain reaction-based DNA sequencing of the 13 most common genes (KAL1, GNRHR, FGFR1, KISS1R, TAC3, TACR3, FGF8, PROKR2, PROK2, CHD7, NELF, GNRH1, and WDR11). MAIN OUTCOME MEASURE(S): The identification of mutations absent in ≥188 ethnically matched controls. Both SIFT (sorting intolerant from tolerant) and conservation among orthologs provided supportive evidence for pathologic roles. RESULT(S): In group 1, 6 (25%) of 24 IHH/KS patients had a heterozygous mutation in a second gene, and in group 2, 13 (54.2%) of 24 had a mutation in at least one gene, but none had digenic mutations. In group 2, 7 (29.2%) of 24 had a mutation considered sufficient to cause the phenotype. CONCLUSION(S): When the 13 most common IHH/KS genes are studied, the overall prevalence of digenic gene mutations in IHH/KS was 12.5%. In addition, approximately 30% of patients without a known mutation had a mutation in a single gene. With the current state of knowledge, these findings suggest that most IHH/KS patients have a monogenic etiology.