Ravikumar Balasubramanian

Prioritizing genetic testing in patients with Kallmann syndrome using clinical phenotypes.

CONTEXT The complexity of genetic testing in Kallmann syndrome (KS) is growing and costly. Thus, it is important to leverage the clinical evaluations of KS patients to prioritize genetic screening. OBJECTIVE The objective of the study was to determine which reproductive and nonreproductive phenotypes of KS subjects have implications for specific gene mutations. SUBJECTS Two hundred nineteen KS patients were studied: 151 with identified rare sequence variants (RSVs) in 8 genes known to cause KS (KAL1, NELF, CHD7, HS6ST1, FGF8/FGFR1, or PROK2/PROKR2) and 68 KS subjects who remain RSV negative for all 8 genes. MAIN OUTCOME MEASURES Reproductive and nonreproductive phenotypes within each genetic group were measured. RESULTS Male KS subjects with KAL1 RSVs displayed the most severe reproductive phenotype with testicular volumes (TVs) at presentation of 1.5 ± 0.1 mL vs 3.7 ± 0.3 mL, P < .05 vs all non-KAL1 probands. In both sexes, synkinesia was enriched but not unique to patients with KAL1 RSVs compared with KAL1-negative probands (43% vs 12%; P < .05). Similarly, dental agenesis and digital bone abnormalities were enriched in patients with RSVs in the FGF8/FGFR1 signaling pathway compared with all other gene groups combined (39% vs 4% and 23% vs 0%; P < .05, respectively). Hearing loss marked the probands with CHD7 RSVs (40% vs 13% in non-CHD7 probands; P < .05). Renal agenesis and cleft lip/palate did not emerge as statistically significant phenotypic predictors. CONCLUSIONS Certain clinical features in men and women are highly associated with genetic causes of KS. Synkinesia (KAL1), dental agenesis (FGF8/FGFR1), digital bony abnormalities (FGF8/FGFR1), and hearing loss (CHD7) can be useful for prioritizing genetic screening.

Human GnRH Deficiency: A Unique Disease Model to Unravel the Ontogeny of GnRH Neurons

Evolutionary survival of a species is largely a function of its reproductive fitness. In mammals, a sparsely populated and widely dispersed network of hypothalamic neurons, the gonadotropin-releasing hormone (GnRH) neurons, serve as the pilot light of reproduction via coordinated secretion of GnRH. Since it first description, human GnRH deficiency has been recognized both clinically and genetically as a heterogeneous disease. A spectrum of different reproductive phenotypes comprised of congenital GnRH deficiency with anosmia (Kallmann syndrome), congenital GnRH deficiency with normal olfaction (normosmic idiopathic hypogonadotropic hypogonadism), and adult-onset hypogonadotropic hypogonadism has been described. In the last two decades, several genes and pathways which govern GnRH ontogeny have been discovered by studying humans with GnRH deficiency. More importantly, detailed study of these patients has highlighted the emerging theme of oligogenicity and genotypic synergism, and also expanded the phenotypic diversity with the documentation of reversal of GnRH deficiency later in adulthood in some patients. The underlying genetic defect has also helped understand the associated nonreproductive phenotypes seen in some of these patients. These insights now provide practicing clinicians with targeted genetic diagnostic strategies and also impact on clinical management.

Olfactory Phenotypic Spectrum in Idiopathic Hypogonadotropic Hypogonadism: Pathophysiological and Genetic Implications

CONTEXT The olfactory phenotype in patients with idiopathic hypogonadotropic hypogonadism (IHH) ranges from complete anosmia (Kallmann syndrome) to normosmia (normosmic IHH). However, the true prevalence of intermediary olfactory phenotypes (hyposmia) in IHH patients has not yet been assessed, and systematic correlations with anatomical and genetic abnormalities have not been reported. OBJECTIVE The objective of this study was to evaluate olfactory function in a large IHH cohort and correlate these findings with olfactory magnetic resonance imaging (MRI) and underlying genetic etiology. DESIGN AND SETTING We conducted a cross-sectional case-control study at an academic referral center. PATIENTS A total of 286 IHH patients (201 males and 85 females) and 2183 healthy historic controls (1011 males and 1172 females) were studied. MAIN OUTCOME MEASURES We measured olfactory function using the University of Pennsylvania Smell Identification Test; in 208 subjects, the genetic etiology of IHH was ascertained by DNA sequencing; in a minor subset [39 of 286 subjects (13%)], olfactory structures were determined by MRI. RESULTS In the IHH cohort, 31.5% were anosmic, 33.6% were hyposmic, and 34.9% were normosmic. Most hyposmic (seven of 11) subjects with MRI data exhibited olfactory structure abnormalities. Of hyposmic subjects, 39.5% harbored mutations in genes involved in either GnRH neuronal migration or GnRH secretion. CONCLUSIONS IHH subjects display a broad spectrum of olfactory function, with a significant hyposmic phenotype in nearly one third of subjects. The hyposmic subjects harbor mutations in genes affecting GnRH neuronal migration and its secretion, suggesting a pathophysiological overlap between Kallmann syndrome and normosmic IHH. Accurate olfactory phenotyping in IHH subjects will inform the pathophysiology of this condition and guide genetic testing.

Isolated GnRH Deficiency: A Disease Model Serving as a Unique Prism into the Systems Biology of the GnRH Neuronal Network

1. Normal development of the reproductive axis activity in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. Isolated GnRH deficiency in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1. Phenotypic features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1.1. Olfactory function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1.2. The clinical spectrum of reproductive defects in humans with isolated GnRH deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1.3. Time of onset of reproductive deficit and duration of reproductive failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1.4. Patterns of GnRH secretory defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.1.5. Non-reproductive phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3. An evolving understanding of the genetic architecture of GnRH deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1. Only half of the genes causing isolated GnRH deficiency have been identified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2. Monogenic vs. oligogenic genetic architecture to human GnRH deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.3. Epigenetic interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

An ancient founder mutation in PROKR2 impairs human reproduction

Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutations age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.

Reversal of isolated hypogonadotropic hypogonadism: long-term integrity of hypothalamo-pituitary-testicular axis in two men is dependent on intermittent androgen exposure

Fawzi Bakiri*, Javier Herrera†, Maria Riestra‡, Olivier Perrichot*, Joaquin Pertierra§, Oscar Domingo Bruno†, Thierry Brue¶** and Frederic Castinetti¶** *Department of Endocrinology, Centre Hospitalier Departemental Felix Guyon, St Denis, France, †Division of Endocrinology, Hospital de Cl ınicas, University of Buenos Aires, Buenos Aires, Argentina, ‡Endocrinology Unit, Hospital de Cabue~ nes, Gij on, §Hospital Universitario Central de Asturias, Oviedo, Spain, ¶Department of Endocrinology, Aix-Marseille University, Reference Center for Rare Pituitary Diseases, La Timone Hospital, Assistance Publique Hôpitaux de Marseille, **Centre de Recherches en Neurobiologie et Neurophysiologie de Marseille (CRN2M), CNRS UMR7286, Marseille, France E-mail: frederic.castinetti@ap-hm.fr

Combination peroxisome proliferator-activated receptor γ and α agonist treatment in Type 2 diabetes prevents the beneficial pioglitazone effect on liver fat content

Diabet. Med. 27, 150–156 (2010)

Expanding the Spectrum of Founder Mutations Causing Isolated Gonadotropin-Releasing Hormone Deficiency.

CONTEXT Loss of function (LoF) mutations in more than 20 genes are now known to cause isolated GnRH deficiency (IGD) in humans. Most causal IGD mutations are typically private, ie, limited to a single individual/pedigree. However, somewhat paradoxically, four IGD genes (GNRH1, TAC3, PROKR2, and GNRHR) have been shown to harbor LoF founder mutations that are shared by multiple unrelated individuals. It is not known whether similar founder mutations occur in other IGD genes. OBJECTIVE The objective of the study was to determine whether shared deleterious mutations in IGD-associated genes represent founder alleles. SETTING This study was an international collaboration among academic medical centers. METHODS IGD patients with shared mutations, defined as those documented in three or more unrelated probands in 14 IGD-associated genes, were identified from various academic institutions, the Human Gene Mutation Database, and literature reports by other international investigators. Haplotypes of single-nucleotide polymorphisms and short tandem repeats surrounding the mutations were constructed to assess genetic ancestry. RESULTS A total of eight founder mutations in five genes, GNRHR (Q106R, R262Q, R139H), TACR3 (W275X), PROKR2 (R85H), FGFR1 (R250Q, G687R), and HS6ST1 (R382W) were identified. Most founder alleles were present at low frequency in the general population. The estimated age of these mutant alleles ranged from 1925 to 5600 years and corresponded to the time of rapid human population expansion. CONCLUSIONS We have expanded the spectrum of founder alleles associated with IGD to a total of eight founder mutations. In contrast to the approximately 9000-year-old PROKR2 founder allele that may confer a heterozygote advantage, the rest of the founder alleles are relatively more recent in origin, in keeping with the timing of recent human population expansion and any selective heterozygote advantage of these alleles requires further evaluation.

Absence of Central Circadian Pacemaker Abnormalities in Humans With Loss of Function Mutation in Prokineticin 2

CONTEXT Loss of prokineticin 2 (PROK2) signaling in mice disrupts circadian rhythms, but the role of PROK2 signaling in the regulation of circadian rhythms in humans is undetermined. OBJECTIVE The aim of the study was to examine the circadian rhythms of humans with a complete loss-of-function PROK2 mutation using an inpatient constant routine (CR) protocol. DESIGN AND SETTING We conducted a case study in an academic medical center. SUBJECTS AND METHODS Two siblings (one male and one female, ages 67 and 62 y, respectively) with isolated GnRH deficiency (IGD) due to a biallelic loss-of-function PROK2 mutation were studied using an inpatient CR protocol. Historical data from inpatient CR protocols conducted in healthy controls (ages 65-81 y) were used for comparison. MAIN OUTCOME MEASURES We measured circadian phase markers (melatonin, cortisol, and core body temperature) and neurobehavioral performance (psychomotor vigilance task [PVT] and subjective alertness scale). RESULTS Circadian waveforms of melatonin and cortisol did not differ between the IGD participants with PROK2 mutation and controls. In both IGD participants, neurobehavioral testing with PVT showed disproportionate worsening of PVT lapses and median reaction time in the second half of the CR. CONCLUSIONS Humans with loss of PROK2 signaling lack abnormalities in circadian phase markers, indicating intact central circadian pacemaker activity in these patients. These results suggest that PROK2 signaling in humans is not required for central circadian pacemaker function. However, impaired PVT in the PROK2-null participants despite preserved endocrine rhythms suggests that PROK2 may transmit circadian timing information to some neurobehavioral neural networks.