Rare structural variants, aneuploidies, and mosaicism in individuals with Mullerian aplasia detected by optical genome mapping.

The molecular basis of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome remains largely unknown. Pathogenic variants in WNT4 and HNF1B have been confirmed in a small percent of individuals. A variety of copy number variants have been reported, but causal gene(s) remain to be identified. We hypothesized that rare structural variants (SVs) would be present in some individuals with MRKH, which could explain the genetic basis of the syndrome. Large molecular weight DNA was extracted from lymphoblastoid cells from 87 individuals with MRKH and available parents. Optical genome mapping (OGM) was performed to identify SVs, which were confirmed by another method (quantitative PCR, chromosomal microarray, karyotype, or fluorescent in situ hybridization) when possible. Thirty-four SVs that overlapped coding regions of genes with potential involvement in MRKH were identified, 14 of which were confirmed by a second method. These 14 SVs were present in 17/87 (19.5%) of probands with MRKH and included seven deletions, three duplications, one new translocation in 5/50 cells-t(7;14)(q32;q32), confirmation of a previously identified translocation-t(3;16)(p22.3;p13.3), and two aneuploidies. Of interest, three cases of mosaicism (3.4% of probands) were identified-25% mosaicism for trisomy 12, 45,X(75%)/46,XX (25%), and 10% mosaicism for a 7;14 translocation. Our study constitutes the first systematic investigation of SVs by OGM in individuals with MRKH. We propose that OGM is a promising method that enables a comprehensive investigation of a variety of SVs in a single assay including cryptic translocations and mosaic aneuploidies. These observations suggest that mosaicism could play a role in the genesis of MRKH.

Biallelic variants in WARS1 cause a highly variable neurodevelopmental syndrome and implicate a critical exon for normal auditory function.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.

Genetics of agenesis/hypoplasia of the uterus and vagina: narrowing down the number of candidate genes for Mayer-Rokitansky-Küster-Hauser Syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome consists of congenital absence of the uterus and vagina and is often associated with renal, skeletal, cardiac, and auditory defects. The genetic basis is largely unknown except for rare variants in several genes. Many candidate genes have been suggested by mouse models and human studies. The purpose of this study was to narrow down the number of candidate genes. METHODS: Whole exome sequencing was performed on 111 unrelated individuals with MRKH; variant analysis focused on 72 genes suggested by mouse models, human studies of physiological candidates, or located near translocation breakpoints in t(3;16). Candidate variants (CV) predicted to be deleterious were confirmed by Sanger sequencing. RESULTS: Sanger sequencing verified 54 heterozygous CV from genes identified through mouse (13 CV in 6 genes), human (22 CV in seven genes), and translocation breakpoint (19 CV in 11 genes) studies. Twelve patients had ≥ 2 CVs, including four patients with two variants in the same gene. One likely digenic combination of LAMC1 and MMP14 was identified. CONCLUSION: We narrowed 72 candidate genes to 10 genes that appear more likely implicated. These candidate genes will require further investigation to elucidate their role in the development of MRKH.

Twenty-year follow-up of the facial phenotype of Brazilian patients with Sotos syndrome.

Sotos syndrome is characterized by overgrowth starting before birth through childhood with intellectual disability and craniofacial anomalies. The majority of patients are large for gestational age with developmental delay or intellectual disability. The majority of cases are caused by pathogenic variants in NSD1. The most consistent physical features in this disorder are facial dysmorphisms including prominent forehead, downslanted palpebral fissures, prognathism with a pointed chin, and a long and narrow face. We present a follow-up to a cohort of 11 individuals found to harbor heterozygous, pathogenic, or likely pathogenic variants in NSD1. We analyzed the facial dysmorphisms and the condition using retrospective over 20 years. Among these patients, followed in our medical genetics outpatient clinic for variable periods of time, all had a phenotype compatible with the characteristic Sotos syndrome facial features, which evolved with time and became superimposed with natural aging modifications. We present here a long-term follow-up of facial features of Brazilian patients with molecularly confirmed Sotos syndrome. In this largest Brazilian cohort of molecularly confirmed patients with Sotos syndrome to date, we provide a careful description of the facial phenotype, which becomes less pronounced with aging and possibly more difficult to recognize in adults. These results may have broad clinical implications for diagnosis and add to the global clinical delineation of this condition.

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.).

Identification of Rare Genetic Variants in the PCDH Genetic Family in a Cohort of Transgender Women.

OBJECTIVE: To study the identification of rare genetic variants in the PCDH genetic family in a cohort of transgender women and their potential role in gender identity. DESIGN: Exome sequencing and functional ontology analysis. SETTING: Augusta University, including the Equality Clinic of Augusta and the Reproductive Medicine and Infertility Associates Clinic. PATIENTS: 24 transgender women and 22 cisgender men. INTERVENTIONS: Exome sequencing followed by variant confirmation through Sanger sequencing and functional classification analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. MAIN OUTCOME MEASURES: Identification of rare, functionally significant genetic variants in the PCDH gene family and their prevalence in transgender women compared to cisgender men. RESULTS: Exome sequencing revealed 38,524 genetic variants, of which 2441 were rare and predicted to be functionally significant. DAVID analysis demonstrated a statistically enriched functional group, "homophilic cell adhesion via plasma membrane adhesion molecules" (Benjamini corrected p-value 1.5 x 10-11), containing 55 genes, including 18 PCDH gene family members. A total of 37 rare variants in 21 PCDH genes were identified, with 36 confirmed by Sanger sequencing. A statistically significant increase in these variants was observed in transgender women compared to cisgender men (Z = 2.08905, p= 0.037). CONCLUSIONS: Transgender women exhibited a greater than 3-fold increase in functionally significant PCDH gene variants compared to cisgender men. These findings suggest that the PCDH family may play a role in the genetic pathways associated with gender identity in transgender women.

Prevalence of pathogenic variants and digenic disease in patients diagnosed with normosmic hypogonadotropic hypogonadism/Kallmann Syndrome.

BACKGROUND: Hypogonadotropic hypogonadism (HH) is due to impaired gonadotropin releasing hormone (GnRH) action resulting in absent puberty and infertility. At least 44 genes have been identified to possess genetic variants in 40-50% of nHH/KS, and 2-20% have presumed digenic disease, but not all variants have been characterized in vitro. HYPOTHESIS: The prevalence of pathogenic (P)/likely pathogenic (LP) variants in monogenic and digenic nHH/KS is lower than reported. DESIGN: Cross-sectional study. SETTING: University Research Laboratory. SUBJECTS: 158 patients with nHH/KS. METHODS: Exome sequencing (ES) was performed and variants were filtered for 44 known genes using Varsome and confirmed by Sanger Sequencing. MAIN OUTCOME MEASURES: P/LP variants in nHH/KS genes. RESULTS: ES resulted in >370,000 variants, from which variants in 44 genes were filtered. Thirty-one confirmed P/LP variants in 10 genes (ANOS1, CHD7, DUSP6, FGFR1, HS6ST1, KISS1, PROKR2, SEMA3A, SEMA3E, TACR3), sufficient to cause disease, were identified in 30/158 (19%) patients. Only 2/158 (1.2%) patients had digenic variant combinations: a male with hemizygous ANOS1 and heterozygous TACR3 variants and a male with heterozygous SEMA3A and SEMA3E variants. Two patients (1.2%) had compound heterozygous GNRHR (autosomal recessive) variants-one P and one variant of uncertain significance (VUS). Five patients (3.2%) had heterozygous P/LP variants in either GNRHR or TACR3 (both autosomal recessive), but no second variant. CONCLUSION: Our prevalence of P/LP variants in nHH/KS was 19%, and digenicity was observed in 1.2%. These findings are less than those previously reported, and probably represent a more accurate estimation since VUS are not included.

Heterozygous ZNHIT3 variants within the 17q12 recurrent deletion region are associated with Mayer-Rokitansky-Kuster Hauser (MRKH) syndrome.

The molecular basis of mullerian aplasia, also known as Mayer-Rokitansky-Kuster Hauser (MRKH) or congenital absence of the uterus and vagina, is largely unknown. We applied a multifaceted genetic approach to studying the pathogenesis of MRKH including exome sequencing of trios and duos, genome sequencing of families, qPCR, RT-PCR, and Sanger sequencing to detect intragenic deletions, insertions, splice variants, single nucleotide variants, and rearrangements in 132 persons with MRKH. We identified two heterozygous variants in ZNHIT3 localized to a commonly involved CNV region at chromosome 17q12 in two different families with MRKH. One is a frameshift, truncating variant that is predicted to interfere with steroid hormone binding of the LxxLL sequence of the C-terminal region. The second variant is a double missense/stopgain variant. Both variants impair protein expression in vitro. In addition, four more probands with MRKH harbored the stopgain variant without the nearby missense variant. In total, 6/132 (4.5%) of patients studied, including five with associated anomalies (type 2 MRKH), had ZNHIT3 variants that impair function in vitro. Our findings implicate ZNHIT3 as an important gene associated with MRKH within the 17q12 CNV region.

Investigation of subfertility in the female Nsmf knockout mouse.

OBJECTIVE: To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. DESIGN: Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. SETTING: University research laboratory. PATIENTS: None; mice were used. INTERVENTIONS: Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. MAIN OUTCOME MEASURES: Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. RESULTS: We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. CONCLUSIONS: Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Long-Term Follow-Up and Treatment of a Female With Complete Estrogen Insensitivity.

CONTEXT: We previously reported the first female with a causative ESR1 gene variant, who exhibited absent puberty and high estrogens. At age 15 years, she presented with lower abdominal pain, absent breast development, primary amenorrhea, and multicystic ovaries. The natural history of complete estrogen insensitivity (CEI) in women is unknown. OBJECTIVE: The purpose of this report is to present the neuroendocrine phenotype of CEI, identify potential ligands, and determine the effect of targeted treatment. DESIGN: We have characterized gonadotropin pulsatility and followed this patient's endocrine profile and bone density over 8 years. Seventy-five different compounds were tested for transactivation of the variant receptor. A personalized medicine approach was tailored to our patient. SETTING: Academic medical center. PATIENT OR OTHER PARTICIPANTS: A 24-year-old adopted white female with CEI. INTERVENTION(S): The patient was treated with diethylstilbestrol (DES) for approximately 2.5 years. MAIN OUTCOME MEASURE(S): Induction of secondary sexual characteristics. RESULTS: Luteinizing hormone (LH) pulse studies demonstrated normal pulsatile LH secretion, elevated mean LH, and mildly elevated mean follicle-stimulating hormone (FSH) in the presence of markedly increased estrogens. DES transactivated the variant ESR1 in vitro. However, DES treatment did not induce secondary sexual characteristics in our patient. CONCLUSIONS: Treatment with DES was not successful in our patient. She remains hypoestrogenic despite the presence of ovarian cysts with a hypoestrogenic vaginal smear, absent breast development, and low bone mineral mass. Findings suggest additional receptor mechanistic actions are required to elicit clinical hormone responses.

The Use of Whole Exome Sequencing in a Cohort of Transgender Individuals to Identify Rare Genetic Variants.

Approximately 0.5-1.4% of natal males and 0.2-0.3% of natal females meet DSM-5 criteria for gender dysphoria, with many of these individuals self-describing as transgender men or women. Despite recent improvements both in social acceptance of transgender individuals as well as access to gender affirming therapy, progress in both areas has been hampered by poor understanding of the etiology of gender dysphoria. Prior studies have suggested a genetic contribution to gender dysphoria, but previously proposed candidate genes have not yet been verified in follow-up investigation. In this study, we expand on the topic of gender identity genomics by identifying rare variants in genes associated with sexually dimorphic brain development and exploring how they could contribute to gender dysphoria. To accomplish this, we performed whole exome sequencing on the genomic DNA of 13 transgender males and 17 transgender females. Whole exome sequencing revealed 120,582 genetic variants. After filtering, 441 variants in 421 genes remained for further consideration, including 21 nonsense, 28 frameshift, 13 splice-region, and 225 missense variants. Of these, 21 variants in 19 genes were found to have associations with previously described estrogen receptor activated pathways of sexually dimorphic brain development. These variants were confirmed by Sanger Sequencing. Our findings suggest a new avenue for investigation of genes involved in estrogen signaling pathways related to sexually dimorphic brain development and their relationship to gender dysphoria.

The prevalence of intragenic deletions in patients with idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

Idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) are clinically and genetically heterogeneous disorders caused by a deficiency of gonadotrophin-releasing hormone (GnRH). Mutations in three genes--KAL1, GNRHR and FGFR1--account for 15-20% of all causes of IHH/KS. Nearly all mutations are point mutations identified by traditional PCR-based DNA sequencing. The relatively new method of multiplex ligation-dependent probe amplification (MLPA) has been successful for detecting intragenic deletions in other genetic diseases. We hypothesized that MLPA would detect intragenic deletions in approximately 15-20% of our cohort of IHH/KS patients. Fifty-four IHH/KS patients were studied for KAL1 deletions and 100 were studied for an autosomal panel of FGFR1, GNRH1, GNRHR, GPR54 and NELF gene deletions. Of all male and female subjects screened, 4/54 (7.4%) had KAL1 deletions. If only anosmic males were considered, 4/33 (12.1%) had KAL1 deletions. No deletions were identified in any of the autosomal genes in 100 IHH/KS patients. We believe this to be the first study to use MLPA to identify intragenic deletions in IHH/KS patients. Our results indicate approximately 12% of KS males have KAL1 deletions, but intragenic deletions of the FGFR1, GNRH1, GNRHR, GPR54 and NELF genes are uncommon in IHH/KS.

JAK/STAT signaling pathway gene expression is reduced following Nelf knockdown in GnRH neurons.

Hypothalamic gonadotropin releasing hormone (GnRH) is crucial for the proper function of the hypothalamic-pituitary-gonadal (HPG) axis, subsequent puberty, and reproduction. When GnRH neuron migration or GnRH regulation is impaired, hypogonadotropic hypogonadism results. Mutations in the gene for nasal embryonic luteinizing hormone-releasing factor (NELF) have been identified in GnRH-deficient humans. NELF is a predominantly nuclear protein that may participate in gene transcription, but the genes NELF regulates are unknown. To address this question, RNA was extracted from NLT GnRH neuronal cells following either stable Nelf knockdown or scrambled control and subjected to cDNA arrays. Transcription factors and cell migration gene expression was altered most commonly. Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, including Stat1, Stat2, Stat5a, Jak2, Irf7 and Irf9, were significantly down regulated as assessed by RT-qPCR. Protein levels of STAT1, phospho-STAT1, and JAK2 were reduced, but the protein level of phospho-JAK2 was not. These findings suggest a role for NELF in the regulation of the JAK/STAT signaling pathway, which have important functions in GnRH neurons.

Copy number variation and regions of homozygosity analysis in patients with MÜLLERIAN aplasia.

BACKGROUND: Little is known about the genetic contribution to Müllerian aplasia, better known to patients as Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Mutations in two genes (WNT4 and HNF1B) account for a small number of patients, but heterozygous copy number variants (CNVs) have been described. However, the significance of these CNVs in the pathogenesis of MRKH is unknown, but suggests possible autosomal dominant inheritance. We are not aware of CNV studies in consanguineous patients, which could pinpoint genes important in autosomal recessive MRKH. We therefore utilized SNP/CGH microarrays to identify CNVs and define regions of homozygosity (ROH) in Anatolian Turkish MRKH patients. RESULTS: Five different CNVs were detected in 4/19 patients (21%), one of which is a previously reported 16p11.2 deletion containing 32 genes, while four involved smaller regions each containing only one gene. Fourteen of 19 (74%) of patients had parents that were third degree relatives or closer. There were 42 regions of homozygosity shared by at least two MRKH patients which was spread throughout most chromosomes. Of interest, eight candidate genes suggested by human or animal studies (RBM8A, CMTM7, CCR4, TRIM71, CNOT10, TP63, EMX2, and CFTR) reside within these ROH. CONCLUSIONS: CNVs were found in about 20% of Turkish MRKH patients, and as in other studies, proof of causation is lacking. The 16p11.2 deletion seen in mixed populations is also identified in Turkish MRKH patients. Turkish MRKH patients have a higher likelihood of being consanguineous than the general Anatolian Turkish population. Although identified single gene mutations and heterozygous CNVs suggest autosomal dominant inheritance for MRKH in much of the western world, regions of homozygosity, which could contain shared mutant alleles, make it more likely that autosomal recessively inherited causes will be manifested in Turkish women with MRKH.

Genetic analysis of Mayer-Rokitansky-Kuster-Hauser syndrome in a large cohort of families.

OBJECTIVE: To study the genetic cause of Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). Although a few candidate genes and genomic domains for have been reported for MRKH, the genetic underpinnings remain largely unknown. Some of the top candidate genes are WNT4, HNF1B, and LHX1. The goals of this study were to: 1) determine the prevalence of WNT4, HNF1B, and LHX1 point mutations, as well as new copy number variants (CNVs) in people with MRKH; and 2) identify and characterize MRKH cohorts. DESIGN: Laboratory- and community-based study. SETTING: Academic medical centers. PATIENT(S): A total of 147 MRKH probands and available family members. INTERVENTIONS(S): DNA sequencing of WNT4, HNF1B, and LHX1 in 100 MRKH patients, chromosomal microarray analysis in 31 North American MRKH patients, and characterization and sample collection of 147 North American and Turkish MRKH probands and their families. MAIN OUTCOME MEASURE(S): DNA sequence variants and CNVs; pedigree structural analysis. RESULT(S): We report finding CNVs in 6/31 people (∼19%) with MRKH, but no point mutations or small indels in WNT4, HNF1B, or LHX1 in 100 MRKH patients. Our MRKH families included 43 quads, 26 trios, and 30 duos. Of our MRKH probands, 87/147 (59%) had MRKH type 1 and 60/147 (41%) had type 2 with additional anomalies. CONCLUSION(S): Although the prevalence of WNT4, HNF1B, and LHX1 point mutations is low in people with MRKH, the prevalence of CNVs was ∼19%. Further analysis of our large familial cohort of patients will facilitate gene discovery to better understand the complex etiology of MRKH.

A balanced chromosomal translocation involving chromosomes 3 and 16 in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome reveals new candidate genes at 3p22.3 and 16p13.3.

BACKGROUND: Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, or the congenital absence of uterus and vagina, is the most severe anomaly of the female reproductive tract. It affects 1 in 5,000 females, and is the second most common cause of primary amenorrhea. The etiology remains unknown in most patients, although four single gene defects and some repetitive copy number variants (CNVs) have been identified. Translocations in MRKH patients are very rare, and reported only in three patients previously without breakpoint mapping. We have identified the fourth MRKH translocation patient and are the first to characterize the breakpoints mapped by molecular methods. RESULTS: The proband is a 17- year old white female with agenesis of the uterus and vagina who had a peripheral blood karyotype revealing a de novo balanced translocation 46,XX,t(3;16)(p22.3;p13.3)dn. There were no known related anomalies present in the proband or her family. No CNVs were found by chromosomal microarray analysis, and no genes were directly disrupted by the translocation. DNA sequencing of six nearby candidate genes-TRIM71, CNOT10, ZNF200, OR1F1, ZNF205, and ZNF213-did not reveal any mutations. RT-qPCR of proband lymphoblast RNA for 20 genes near the breakpoints of 3p22.3 and 16p13.3 showed significantly altered expression levels for four genes in the proband compared to three white female controls, after correction for multiple comparisons. Reduced expression was seen for CMTM7 and CCR4 on 3p22.3, while increased expression was observed for IL32 and MEFV on 16p13.3. CONCLUSION: We have mapped the breakpoints of our t(3;16)(p22.3;p13.3) translocation patient using molecular methods to within 13.6 kb at 3p22.3 and within 1.9 kb for 16p13.3 and have suggested 10 nearby genes that become plausible candidate genes for future study.

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.

Long-term follow-up of females with unbalanced X;Y translocations-reproductive and nonreproductive consequences.

BACKGROUND: Females with Xp;Yq translocations manifest short stature and normal fertility, but rarely have follow-up. The study purpose was to define the phenotype of a family with t(X;Y)(p22.3;q11.2), determine long-term reproductive function, and compare to all reported female cases. METHODS: Comprehensive clinical and molecular analyses were performed on the female proband, who had regular menses, normal endocrine function, and three pregnancies spanning seven years--a normal liveborn male and two with unbalanced translocations (liveborn female and stillborn male). RESULTS: The translocation truncated KAL1 and deleted 44 genes on der(X). Our report constitutes the longest follow-up of an X;Y translocation female. She had no evidence of Kallmann syndrome, gonadoblastoma, or cardiovascular disease. Detailed analysis of 50 published female cases indicated a uniform lack of follow-up and significant morbidity-intellectual disability (10%), facial dysmorphism (28%), eye abnormalities (14%), and skeletal defects (28%). CONCLUSIONS: Our findings indicate normal ovarian function to date in a woman with an t(X;Y)(p22.3;q11.2). However, additional published studies in the literature suggest careful follow-up is necessary and contradict the generalization that females with Xp;Yq translocations are usually normal except for short stature.