Genetics of hypopituitarism {#sec1-1}
===========================

Hypopituitarism is an endocrine syndrome characterized by reduced or absent secretion of one or more anterior pituitary hormones with consequent dysfunction of the corresponding peripheral glands. Deficiencies in all the hormones is defined as pan-hypopituitarism, the lack of two or more hormones is called partial hypopituitarism, whereas the absence of a single hormone is defined as selective hypopituitarism. Pan-hypopituitarism is the rarest of the three. Several forms exist: congenital, acquired, organic and functional ([@ref1]).

Combined pituitary hormone deficiency (CPHD) is characterized by impaired production of several pituitary hormones, such as growth hormone, thyroid-stimulating hormone, prolactin, adrenocorticotropic hormone and gonadotropic hormone, and is caused by mutations in transcription factors involved in pituitary ontogenesis. Congenital hypopituitarism has a low incidence with respect to secondary hypopituitarism due to pituitary adenomas, trans-sphenoidal surgery, or radiotherapy. The incidence of congenital hypopituitarism in the population is 1:3000-4000 ([@ref2]). Genetic mutations associated with congenital hypopituitarism mainly affect eight genes encoding transcription factors: *PROP1* (thyroid-stimulating, follicle-stimulating, growth, luteinizing and adrenocorticotropic hormones and prolactin are low or absent), *POU1F1* (growth and thyroid-stimulating hormones and prolactin are low or absent), *HESX1* (thyrotropin, follicle-stimulating, growth, luteinizing and adrenocorticotropic hormones are low or absent), *LHX3* (thyroid-stimulating, follicle-stimulating, growth, luteinizing and adrenocorticotropic hormones and prolactin are low or absent), and *LHX4* (thyroid-stimulating, growth, luteinizing, follicle-stimulating and adrenocorticotropic hormones are low or absent) ([@ref2]).

The clinical phenotype depends on the affected hormone, the severity of pituitary impairment and age of onset. In childhood, congenital idiopathic forms are the most frequent, and are associated with developmental retardation, delay of puberty and absence of adrenarche. In adulthood, acquired forms are more frequent ([@ref3]).

Loss-of-function mutations in *PROP1* are the most common cause of sporadic and familial cases of CPHD. This gene is mutated in 11% of cases. The mutation rate, however, varies considerably in relation to geographical area. The prevalence of the mutation is less than 1% in western European, American, Australian and Japanese populations, and higher in Russian and eastern European populations. Patients with mutations in *PROP1* show growth hormone (GH), prolactin (PL), and thyroid-stimulating hormone (TSH) deficiency and variable defects in the secretion of luteinizing (LH), follicle-stimulating (FSH) and adenocorticotropic (ACTH) hormones ([@ref4]).

Mutations in *POU1F1* are the second most frequent cause of pituitary hormone deficiency. The phenotype associated with *POU1F1* mutations can be inherited by dominant or recessive transmission. The major mutation is the heterozygous p.Arg271Trp, found in \~30% of patients with *POU1F1* mutations. In sporadic cases, mutations in this gene are only found in 1.6% of cases. POU1F1 is a member of the POU family of transcription factors and is expressed in the anterior lobe of the pituitary gland. The phenotype associated with *POU1F1* mutations has severely low levels of GH and PRL, variable levels of TSH, short stature, facial dysmorphism, and dysphagia during infancy ([@ref5]).

Another gene with occasional mutations is *HESX1*. Mutations in this gene occur in 0.45% of sporadic cases. Single heterozygous mutations causes a less severe disorder with incomplete penetrance, whereas homozygous mutations cause a severe and completely penetrant disorder ([@ref6]).

Biallelic mutations in *LHX3* cause deficiencies in GH, PRL, TSH, LH, FSH and ACTH. Mutations in *LHX3* are found in 0.3% of sporadic cases and 11.1% of familial cases ([@ref7]).

The pathological phenotype associated with heterozygous mutations in *LHX4* is inherited as an autosomal dominant trait with variable penetrance. Patients with CPHD have variable reductions in serum levels of GH, TSH, ACTH and gonadotropin. Cranial magnetic resonance imaging shows pituitary gland hypoplasia in most cases. However, there is a wide phenotypic variability within and between families.

Finally, mutations in the *GLI2* gene have been reported in patients with combined pituitary hormone deficiency and ectopic posterior pituitary lobe. For instance, several individuals with truncating mutations in *GLI2* show pituitary anomalies, polydactyly and subtly dysmorphic facial features. The inheritance pattern is dominant with incomplete penetrance and variable phenotype. There are mutations in *GLI2* in 1.5% of CPHD cases.

The genes associated with combined pituitary hormone deficiency are: *PROP1*, *SOX3*, *POU1F1*, *HESX1*, *LHX4*, *LHX3*, *OTX2* and *GLI2* ([Table 1](#T1){ref-type="table"}). Pathogenic variants may be missense, nonsense, splicing or small indels. MAGI uses a multi-gene NGS panel to detect nucleotide variations in coding exons and flanking introns of the genes listed in the table. Our NGS test has an analytical sensitivity (proportion of true positives) and analytical specificity (proportion of true negatives) of ≥99% (coverage depth ≥10x).

###### 

Genes associated with combined pituitary hormone deficiency

  Gene       Inheritance   OMIM gene   OMIM phenotype   OMIM phenotype ID   Gene function
  ---------- ------------- ----------- ---------------- ------------------- --------------------------------------------------------------------------------------------------------------
  *PROP1*    AR            601538      CPHD2            262600              Paired-like homeodomain transcription factor required for pituitary development
  *POU1F1*   AD, AR        173110      CPHD1            613038              Regulation of expression of genes involved in pituitary development and hormone expression
  *HESX1*    AD, AR        601802      CPHD5            182230              Transcriptional repressor expressed in developing forebrain and pituitary gland
  *LHX3*     AR            600577      CPHD3            221750              LIM-containing domain transcription factor required for pituitary development and motor neuron specification
  *LHX4*     AD            602146      CPHD4            262700              LIM-containing domain transcription factor required for pituitary development
  *SOX3*     XLR           313430      PHPX             312000              Transcription factor required for pituitary function and development of CNS midline structures
  *OTX2*     AD            600037      CPHD6            613986              Homeodomain-containing transcription factor required for brain, craniofacial and sensory organ development
  *GLI2*     AD            165230      CJS              615849              Zinc finger transcription factor required for embryogenesis

CJS = Culler-Jones syndrome; CNS = central nervous system; CPHD = combined pituitary hormone deficiency; PHPX = panhypopituitarism, X-linked; AR = autosomal recessive; AD = autosomal dominant; XLR = X-linked recessive.

Primary ciliary dyskinesia {#sec1-2}
==========================

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous group of inherited disorders due to morphological and structural alterations of the cilia. It is characterized by chronic bronchorrhea with bronchiectasis and chronic sinusitis and is the second most common congenital disease of the respiratory system after cystic fibrosis. The prevalence is estimated at around 1:20000 ([@ref8]).

Ultrastructural defects of the 9+2 axoneme of cilia and flagella may be: partial or complete loss of internal dynein arms, central microtubule anomalies, and radial spoke defects. These defects cause recurrent sinusitis, bronchiectasis due to immotile cilia in the upper and lower airways, and infertility due to altered cilia in the oviduct as well as altered sperm flagella ([@ref9]).

Fifty percent of patients show *situs inversus*. The association of *situs inversus,* sinusitis and bronchiectasis is the classical triad known as Kartagener syndrome. It is noteworthy that this syndrome is a subgroup of primary ciliary dyskinesia ([@ref10]). In fact, we know that *situs inversus* is caused by motility failure of nodal cilia that allow lateralization of organs during early embryogenesis ([@ref11]).

In some subjects, primary ciliary dyskinesia is associated with other disorders like polycystic kidney, retinitis pigmentosa, Barder-Biedl syndrome and Usher syndrome, the pathogenesis of which is linked to structural defects of the primary cilia ([@ref12]). Respiratory disorders can appear at birth (neonatal respiratory distress), during infancy and rarely in adulthood, and may include chronic infections of the upper and lower respiratory tract. Bronchiectasis is not present at birth but may be a secondary effect of a chronic lung disease ([@ref8]).

Severity and progression of the disease are variable among patients and depend on what ciliary substructures are altered. About 50% of male patients with PCD are infertile due to lack of sperm motility ([@ref9],[@ref13]). Adult women with PCD may be subfertile and at risk of extra-uterine pregnancies due to delayed oocyte transport through the uterine tubes ([@ref10]). The most frequent ultrastructural defects of PCD in spermatozoa are ([@ref14],[@ref15]):

1.  \- reduction and/or absence of the outer dynein arm: \~38.5% of all PCD cases;

2.  \- reduction and/or absence of both dynein arms (outer and inner): \~10.5% of all PCD cases;

3.  \- microtubule (axoneme) disorganization due to absence of the inner dynein arm and defects in the central apparatus: \~14% of all PCD cases;

4.  \- absence or interruption of central apparatus (i.e. the pair of central microtubules and/or radial spokes): \~7% of all PCD cases;

5.  \- reduction and/or absence of the inner dynein arm (rare);

6.  \- oligocilia with or without normal ultrastructure (rare).

Most cases of primary ciliary dyskinesia or Kartagener syndrome have autosomal recessive inheritance, although some cases with X-linked recessive inheritance have been reported. Currently, 39 genes are known to be involved in PCD ([Table 2](#T2){ref-type="table"}). The most frequent mutations are in: *DNAH5*, *DNAH11*, *CCDC39*, *DNAI1*, *CCDC40*, *CCDC103*, *SPAG1*, *ZMYND10*, *ARMC4*, *CCDC151*, *DNAI2*, *RSPH1*, *CCDC114*, *RSPH4A*, *DNAAF1*, *DNAAF2* and *LRRC6.* [Table 2](#T2){ref-type="table"} shows the frequencies of biallelic pathogenic variants in affected unrelated subjects. Pathogenic variants may be missense, nonsense, splicing and small indels. MAGI uses a multi-gene NGS panel to detect nucleotide variations in coding exons and flanking introns of the genes listed in [Table 2](#T2){ref-type="table"}.

###### 

Genes associated with primary ciliary dyskinesia

  Gene         Inheritance   OMIM gene   OMIM phenotype   OMIM or HGMD phenotype ID   Frequency of biallelic variants in affected unrelated subjects ([@ref22])
  ------------ ------------- ----------- ---------------- --------------------------- ---------------------------------------------------------------------------
  *DNAI1*      AR            604366      CILD1            244400                      2%-10%
  *DNAAF3*     AR            614566      CILD2            606763                      \<1%
  *DNAH5*      AR            603335      CILD3            608644                      15%-29%
  *HYDIN*      AR            610812      CILD5            608647                      \<1%
  *NME8*       AR            607421      CILD6            610852                      \<1%
  *DNAH11*     AR            603339      CILD7            611884                      6%-9%
  *DNAI2*      AR            605483      CILD9            612444                      2%
  *DNAAF2*     AR            612517      CILD10           612518                      \<1%-2%
  *RSPH4A*     AR            612647      CILD11           612649                      1%-2%
  *RSPH9*      AR            612648      CILD12           612650                      \<1%
  *DNAAF1*     AR            613190      CILD13           613193                      1%-2%
  *CCDC39*     AR            613798      CILD14           613807                      4%-9%
  *CCDC40*     AR            613799      CILD15           613808                      3%-4%
  *DNAL1*      AR            610062      CILD16           614017                      \<1%
  *CCDC103*    AR            614677      CILD17           614679                      \<4%
  *DNAAF5*     AR            614864      CILD18           614874                      \<1%
  *LRRC6*      AR            614930      CILD19           614935                      1%
  *CCDC114*    AR            615038      CILD20           615067                      \<2%
  *DRC1*       AR            615288      CILD21           615294                      \<1%
  *ZMYND10*    AR            607070      CILD22           615444                      2%-4%
  *ARMC4*      AR            615408      CILD23           615451                      \<3%
  *RSPH1*      AR            609314      CILD24           615481                      2%
  *C21ORF59*   AR            615494      CILD26           615500                      \<1%
  *CCDC65*     AR            611088      CILD27           615504                      \<1%
  *SPAG1*      AR            603395      CILD28           615505                      \<4%
  *CCNO*       AR            607752      CILD29           615872                      \<1%
  *CCDC151*    AR            615956      CILD30           616037                      \<3%
  *CENPF*      AR            600236      STROMS           243605                      \<1%
  *RSPH3*      AR            615876      CILD32           616481                      \<1%
  *GAS8*       AR            605178      CILD33           616726                      /
  *DNAJB13*    AR            610263      CILD34           617091                      /
  *TTC25*      AR            617095      CILD35           617092                      /
  *PIH1D3*     XLR           300933      CILD36           300991                      9.5%
  *DNAH1*      AR            603332      CILD37           617577                      \<1%
  *STK36*      AR            607652      CILD             1147369503                  /

CILD = ciliary dyskinesia, primary; STROMS = Stromme syndrome; AR = autosomal recessive; XLR = X-linked recessive; HGMD = Human Gene Mutation Database (https://portal.biobase-international.com/hgmd/pro/)

Hypogonadotropic hypogonadism {#sec1-3}
=============================

Correct functioning of the hypothalamo-pituitary-gonadal axis is fundamental for differentiation and sexual development during the fetal period and puberty ([@ref16]). Hypogonadotropic hypogonadism (HH) is caused by alterations in this axis. Such alterations cause low serum levels of sex hormones associated with normal or low levels of FSH and LH. The prevalence of HH is 1/8000 newborns ([@ref17]).

Clinically, patients with HH show little or no sexual development, primary amenorrhea (women) and oligoazoospermia (men). Other possible features may be: cleft palate, tooth agenesis, visual impairment, intellectual disability (and other neurological abnormalities), and renal agenesis ([@ref18]).

Hypogonadotropic hypogonadism may be considered isolated when only the gonads are impaired. There are two forms of the isolated HH: Kallmann syndrome (HH associated with anosmia) is caused by defects in embryonic migration of neurons secreting gonadotropin releasing hormone (GnRH); normosmic HH, in which HH is the only symptom and is due to altered signaling, regulation and secretion of GnRH ([@ref19]).

The HH may have autosomal dominant, autosomal recessive or X-linked inheritance.

The first gene variation discovered in cases of HH was in *ANOS1* (or *KAL1*). *ANOS1* encodes an adhesion molecule (anosmin), probably involved in migration of olfactory and GnRH-secreting neurons toward the hypothalamus during embryo development. Hypogonadotropic hypogonadism associated with *ANOS1* mutations has X-linked recessive inheritance, so only males are affected. Besides HH and anosmia, patients with mutations in *ANOS1* show renal agenesis and neurological disorders such as intellectual disability, sensorineural deafness and synkinesis ([@ref20]).

*GNRHR* was the first gene found to have variations in cases of normosmic HH, a disorder with autosomal recessive inheritance. The gene encodes the GnRH receptor, a protein expressed in the pituitary gland. The associated phenotype is highly variable, ranging from very severe (total absence of puberty) to partial or delayed pubertal development ([@ref21]).

Since involvement of *ANOS1* and *GNRHR* in hypogonadotropic hypogonadism was discovered, 28 other associated-genes have emerged ([Table 3](#T3){ref-type="table"}). More than 2% of cases have mutations in *ANOS1*, *CHD7*, *FGFR1*, *GNRHR*, *IL17RD*, *PROKR2*, *SOX10* or *TACR3*. The other genes have only been found in a few families ([@ref18]).

###### 

Genes associated with hypogonadotropic hypogonadism

  Gene        Inheritance   OMIM gene   OMIM phenotype   OMIM or HGMD phenotype ID   Gene function
  ----------- ------------- ----------- ---------------- --------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------
  *KISS1*     AR            603286      HH13             614842                      Stimulation of GnRH-induced gonadotropin secretion, activation of GnRH neurons
  *HS6ST1*    AD            604846      HH15             614880                      Neuron development, neuron branching
  *IL17RD*    AD, AR        606807      HH18             615267                      Fate-specification of GnRH-secreting neurons
  *PROK2*     AD            607002      HH4              610628                      Chemoattractant for neuronal precursor cells in olfactory bulb
  *GNRHR*     AR            138850      HH7              146110                      Receptor for GnRH. Stimulation of LH and FSH secretion
  *TACR3*     AR            162332      HH11             614840                      Receptor for neurokinin B. Expressed in hippocampus, hypothalamus, substantia nigra
  *SPRY4*     AD            607984      HH17             615266                      Regulation of neurite outgrowth in hippocampal neurons
  *SEMA3A*    AD            603961      HH16             614897                      Inhibition of axonal outgrowth, stimulation of apical dendrite growth
  *FEZF1*     AR            613301      HH22             616030                      Embryonic migration of GnRH-releasing neurons into brain
  *FGF17*     AD            603725      HH20             615270                      Induction and patterning of embryonic brain
  *GNRH1*     AR            152760      HH12             614841                      Stimulation of LH and FSH secretion
  *FGFR1*     AD            136350      HH2              147950                      Mesoderm patterning, correct axial organization during embryo development, skeletogenesis, development of GnRH neuronal system
  *CHD7*      AD            608892      HH5              612370                      Formation of neural crest
  *NSMF*      AD            608137      HH9              614838                      Guidance of olfactory axon projections, migration of LHRH neurons
  *FGF8*      AD            600483      HH6              612702                      Regulation of embryo development, cell proliferation, differentiation, migration. Brain, eye, ear, limb, GnRH neuronal system, hippocampal neuron development
  *WDR11*     AD            606417      HH14             614858                      Regulation of GnRH production
  *FSHB*      AR            136530      HH24             229070                      Beta subunit of FSH. Induction of egg and sperm production
  *TAC3*      AR            162330      HH10             614839                      Central regulator of gonad function
  *DUSP6*     AD            602748      HH19             615269                      Expression regulated by GnRH
  *KISS1R*    AR            604161      HH8              614837                      Neuroendocrine control of gonadotropin axis
  *LHB*       AR            152780      HH23             228300                      Promotion of spermatogenesis and ovulation by stimulating gonads to synthesize steroids
  *PROKR2*    AD            607123      HH3              244200                      Induction of tangential and radial migration of olfactory bulb interneurons
  *FLRT3*     AD            604808      HH21             615271                      Spatial organization of brain neurons.
  *ANOS1*     XLR           300836      HH1 (KS)         308700                      Neural cell adhesion, axonal migration, patterning of mitral and tufted cell collaterals to olfactory cortex
  *SOX10*     AD            602229      WS2E             611584                      Development of neural crest, peripheral nervous system, glia
  *AXL*       AD            109135      HH               1734393901                  GnRH neuron survival and migration
  *CCDC141*   AR            616031      KS               817012261                   Neural radial migration
  *SEMA3E*    AD            608166      KS               817012261                   Ensuring synapse formation specificity
  *SRA1*      AR            603819      HH               1734393901                  Mediation of transcriptional co-activation of steroid receptors

GnRH = gonadotropin-releasing hormone; HH = hypogonadotropic hypogonadism; KS = Kallmann syndrome; WS = Waardenburg syndrome; AD = autosomal dominant; AR = autosomal recessive; XLR = X-linked recessive; HGMD = Human Gene Mutation Database (https://portal.biobase-international.com/hgmd/pro/)

Pathogenic variants may be missense, nonsense, splicing or small indels. MAGI uses a multi-gene NGS panel to detect nucleotide variations in coding exons and flanking introns of the above genes ([Table 3](#T3){ref-type="table"}).

Conclusions {#sec1-4}
===========

We created a NGS panel to detect nucleotide variations in coding exons and flanking regions of all the genes associated with infertility. When a suspect of syndromic infertility is present we perform the analysis of all the genes present in this short article. In order to have a high diagnostic yield, we developed a NGS test that reaches an analytical sensitivity (proportion of true positives) and an analytical specificity (proportion of true negatives) of ≥99% (coverage depth ≥10x).

Conflict of interest: {#sec1-5}
=====================

Each author declares that he or she has no commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangement etc.) that might pose a conflict of interest in connection with the submitted article
