Additional features


Permanent insulin-requiring neonatal diabetes in homozygotes

Low renal threshold for glucose resulting in glycosuria

Pancreatic agenesis and permanent insulin-requiring neonatal diabetes in homozygotes

Renal cysts, progressive non-diabetic renal dysfunction leading to renal failure, genital abnormalities in females


The first GH1 gene defect was identified in an IGHD II kindred that showed segregation with a marker tightly linked to the GH1 gene (Table 1) (19). DNA sequence analysis of PCR amplification products of four affected relatives showed a change in the sixth base of the IVS3 5' SS (Table 1). This mutation causes skipping or deletion of exon 3, which codes for amino acids 32-71, including one of the four cysteines in GH necessary for intramolecular disulfide bonding. The resulting truncated GH protein product retains the signal peptide and exon 4-5 sequences thought to be important for transport to the secretory granules. The presence of the mutant and normal GH protein products within the secretory granules results in derangements and apoptosis of somatotropes that inhibit expression of the normal GH molecule. Recently, the mechanism by which this mutant GH protein interferes with the normal GH protein has been studied in mice transgenic for another mutation (IVS3 + 1 G > A transition) that causes IGHD II. McGuinness et al. found that expression of the mutant protein disrupts secretory vesicles and causes widespread pituitary damage (26).

Two additional IGHD II mutations alter the first base of the IVS3 5' SS. One is a G > C transversion (IVS3 + 1G > C) and the other is a recurring G>A transition (IVS3 + 1G>A) (Table 1) (27,28). The latter was found in three nonrelated families and is thought to be caused by the high mutation frequency at CpG dinucleotides that result in C to T and G to A transitions (discussed further under GHR point mutations). Both IGHD II mutations were proven to cause exon 3 skipping in lymphoblas-toid cells and transfection studies, respectively. Other IGHD II mutations in IVS3 that also cause skipping of exon 3 but do not occur within the branch consensus, 5' SS or 3' SS have been identified (29). The first deletes 18 bp (IVS3 del+ 28-45) and the second is a G > A transition (IVS3 + 28G > A) (Table 1). RT-PCR amplification of GH1 gene transcripts from transient expression studies, using IGHD II mutant constructs in mammalian cells, yielded DNAs showing dramatic increases in exon 3 skipping relative to normal controls. The shift toward exon 3 skipping is caused by the disruption of an intronic XGGG repeat, which is an intron splicing enhancer (ISE) that regulates the pattern of alternative splicing of GH mRNA.

Recently, an additional GH1 mutation was reported that causes IGHD II by its effects on an exon splice enhancer (ESE). Moseley et al. reported an A > G transition of the fifth base of exon 3 (E3 + 5 A > G) in affected individuals from an

IGHD II family. This mutation disrupts a (GAA)n ESE motif immediately following the weak IVS2 3' SS. To determine the effect of ESE mutations on GH mRNA processing, GH3 cells were transfected with expression constructs containing either the normal ESE, +5 A > G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' SS and caused either partial E3 skipping, because of activation of an E3 +45 cryptic 3' SS, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. The authors conclude that the E3 +5 A > G mutation causes IGHD II because it perturbs an ESE required for GH splicing (30).

One IGHD II mutation has been reported that does not occur within IVS3 or affect splicing. This mutation is a G > A transition that results in an Arg > His substitution at residue 183 (Arg183His) of the mature GH protein (Table 1) (31). This substitution is believed to alter the intracellular processing of the normal GH molecules by binding zinc and thereby deranging the zinc-associated presecretory packaging of GH.

3.5.3. X-Linked Isolated Growth Hormone Deficiency A third form of IGHD called IGHD III (OMIM 307200) has an X-linked mode of inheritance and distinct clinical findings in different families. In some families, all cases have agamma-globulinemia associated with their IGHD, whereas in other families, all cases have only IGHD. This suggests that contiguous gene defects on the long arm of the X chromosome might cause some IGHD III cases. Duriez et al. reported that X-linked agammaglobulinemia and IGHD is caused by mutation in the Bruton's tyrosine kinase or BTK gene (32).

Laumonnier et al. studied the SOX3 gene in families with X-linked mental retardation and GH deficiency where the causative gene had been mapped to Xq26-q27. They showed that the SOX3 gene maps to Xq26.3 and was involved in a large family in which affected individuals had mental retardation and IGHD (OMIM 300123 and 313430). The mutation was an inframe duplication of 33 bp encoding 11 alanines in a polyala-nine tract of the SOX3 gene. The expression pattern during neural and pituitary development suggested that dysfunction of the SOX3 gene caused by this polyalanine expansion might disturb transcription pathways and the regulation of genes involved in pituitary development (33).

3.5.4. Biodefective Growth Hormone (Autosomal

Recessive) Some children with short stature comparable to that seen in GH deficiency have low levels of IGF-I but normal levels of GH assayed by radioimmunoassay (RIA) (34,35). In such cases, the administration of rhGH is reported to produce an increase in IGF-I levels and a growth response. In some, the concentration of GH as measured by RIA greatly exceeds the concentration measured by radioreceptor assay. These results suggest that the primary defect could be production of an abnormal GH polypeptide whose alteration causes a reduced somatogenic activity but enables it to react with anti-GH antibodies. Takahashi et al. identified a C > T transition in codon 77 that results in an Arg > Cys substitution in the GH1 gene of a subject diagnosed with bioinactive GH. The patient was heterozygous for the mutation and isoelectric focusing of his serum showed an abnormal GH peak in addition to a normal peak (36). Surprisingly, his father was also heterozygous for the C > T transition but was of normal height and had normal isoelectric focusing results. The disparate findings in the father and his affected child were not explained.

3.5.5. GHRH Receptor Defects Heterogeneous mutations in the human GHRHR gene (OMIM 139191) have been identified. Most patients with these mutations have had poor growth since infancy and were extremely short. They failed to produce GH in response to standard provocative tests and had good responses to GH replacement. A nonsense mutation has been reported in the human GHRHR gene in two first cousins of a consanguineous Indian Moslem family with profound IGHD (37). Both cousins were homozygous for a G > T transversion in exon 3, which converted a Glu to Ter (Glu27Ter) in their GHRHR genes. Subsequently, the same mutation has been identified in an isolate from the Indus valley of Pakistan (38). A second mutation has been identified in a large isolate from Brazil diagnosed with an autosomal recessive form of IGHD. Affected subjects were found to be homozygous for a G > A transition of the first base of IVS1, which is predicted to alter splicing and results in an inactive protein product (39).

3.5.6. Growth Hormone Resistance To be biologically active, GH (OMIM 139250) must bind to its transmembrane receptor GHR (OMIM 600946). GHR then dimerizes, activating intracellular signal transduction pathways resulting in the synthesis and secretion of IGF1 (OMIM 147440). IGF1 binds to its receptor (IGF1R, OMIM 147370) and activates its own signal transduction pathways, resulting in mitogenic and anabolic responses that lead to growth. Disruptions in GHR or IGF1 can cause GH resistance characterized by phenotypic features of GH deficiency associated with normal or high GH levels. Laron Dwarfism I: Growth Hormone Receptor Defects Laron dwarfism is an autosomal recessive disorder caused by GH resistance as a result of defects in GHR (OMIM 600946). Although at the clinical level, Laron syndrome cases might be indistinguishable from GHD cases, they differ at the biochemical level. Patients with Laron dwarfism have low levels of IGF1, despite normal or increased levels of GH. This contrasts with the low levels of both IGF1 and GH that are seen in GHD. Importantly, exogenous GH does not induce an IGF1 response or restore normal growth in Laron dwarfism I cases because of GHR dysfunction. Although the majority of reported patients are Jewish, the disorder has been described in other ethnic groups. Laron dwarfs have the clinical appearance of severe IGHD with very delayed growth, abnormal facial appearance, high-pitched voice, and small male genitalia (41-43). Length at birth might be short in relation to the birth weight, and tooth eruption and fontanelle closure are delayed. Laron dwarfs have the truncal obesity and the increased upper-lower segment body ratios typical of pituitary dwarfs. Although spontaneous hypoglycemia can occur, the production of other anterior pituitary hormones (ACTH, TSH, and gonadotropins) remains intact. Fasting GH levels are usually increased and range from normal to greater than 100 ng/mL. Plasma IGF-I levels are low and, in contrast to those of GH-deficient subjects, do not respond to exogenous rhGH (41,42).

Although plasma levels of the GH-binding proteins (GHBP) that are derived from the extracellular domain of GHR are usually low in Laron dwarfism I cases, Woods et al. reported a homozygous point mutation in the intracellular domain of the GHR that caused Laron syndrome with elevated GHBP levels (44). They predicted that the mutant GHR would not be anchored in the cell membrane but would be measurable in the serum as GHBP, thus explaining the phenotype of severe GH resistance combined with elevated circulating GHBP. Studies of the GHR genes of Laron dwarfism I cases have identified a variety of exon deletions and base substitutions. Although treatment with exogenous GH is ineffective in those with GHR dysfunction, replacement therapy with recombinant IGF1 has been shown to be effective.

Studies indicate that GH produced by Laron dwarfs reacts normally in radioreceptor assays with the GH receptors of normal hepatic cells (42). This, along with their lack of response to exogenous GH, suggests the primary defect might be an abnormality of membrane receptors for GH. Molecular analysis of GH receptor (GHR) genes has shown deletions, point mutations, and splicing defects. GHR Deletions The first examples of GHR mutations reported were deletions of portions of the gene encoding the extracellular domain (45). Southern blotting showed altered restriction patterns of the GHR genes from two of nine patients with Laron syndrome who had no detectable GH-binding protein (GHBP) and very low levels of IGF-I. Although these and other studies were interpreted as showing deletions of exons 3, 5, 6, and part of 4 from the GHR gene, the mechanism by which these two noncontiguous deletions arose remains unclear. GHR Point Mutations Over 50 different mutations in the GHR gene have been reported (see Human Gene Mutation Database, 119984.html). For example, Amselem et al. detected a T > C transition that converts the 96th residue of the extracellular domain from pheny-lalanine to serine (46). Duquesnoy et al. demonstrated that cells transfected with this mutant cDNA lacked GH-binding activity (47).

There are multiple different stop codon mutations of GHR genes in Laron dwarf patients (48). For example, in a patient of northern European origin, a TGC (Cys) to TGA (stop) mutation was detected at codon 38 in exon 4, and a CGA (Arg) to TGA (stop) mutation was found at codon 43 in exon 4 of two

Mediterranean patients who were products of consanguineous marriages. Both stop codons truncate the GHR protein and delete most of its GHBP domain and all of its transmembrane and intracellular domains. These findings are consistent with the lack of GHBP in each of the patients with Laron syndrome. The mechanism of the CGA to TGA mutation is consistent with deamina-tion of 5-methylcytosine that preferentially occurs in CpG dinucleotides. Such dinucleotides often represent "hot spots'' for CG to TG or CG to CA mutations, and 17 occur within the GHR gene. Two of these, at nucleotides 181 and 703, occur in CGA codons that could yield stop codons (46-49). A GAT(Asp) to CAT(His) mutation was identified in exon 6 of two unrelated kindreds with growth deficiency but normal GHBP levels. Conversion of the highly conserved aspartic acid residue to his-tidine was shown to prevent dimerization of the GH receptor which is necessary for GH action (50,51). GHR Splicing Defects Rosenbloom et al. identified 20 patients with Laron syndrome in an inbred population of Spanish extraction in southern Ecuador (52). These patients were -6.7 to -10 SD below the mean height and had limited elbow extension, blue sclera, short limbs, hip degeneration, acrohypoplasia, and normal or superior intelligence. To determine the associated defect in the GHR gene, Berg et al. used denaturing gradient gel electrophoresis to analyze each exon of the GHR gene (53). Unusual fragments derived from exon 6 showed abnormal mobility and DNA sequencing showed an A > G transition in the third position of codon 180, which is 24 nucleotides from the 3' end of exon 6. Although this mutation does not cause an amino acid substitution, it produces a consensus 5' SS sequence within exon 6. The resulting near-consensus 5' SS within exon 6 causes aberrant splicing and deletion of eight amino acids of the 3' end of exon 6. Deletion of these residues is thought to reduce the function of the GHR molecule (53).

Two cousins of Pakistani descent were found to have a G > C transversion at the 5' SS of exon 8 resulting in exon skipping. The mutant protein lacks the transmembrane and intracel-lular domains and results in elevated circulating levels of GH-binding protein in the affected patients (44). Laron Dwarfism II: Post GHR Defects Laron Dwarfism II is caused by post-GHR defects (see OMIM 245590). Patients with Laron dwarfism II have elevated serum GH, normal GHBP levels, and respond well to treatment with IGF-1, indicating their growth deficiency is the result of a post-GHR defect. Woods et al. described a patient with severe growth failure, sensorineural deafness, and mental retardation who was homozygous for a partial deletion of the IGF1 gene (54). RT-PCR analysis confirmed the deletion of exons 4-5 that would result in a severely truncated mature IGF1 peptide. Interestingly, this patient had only a slightly delayed bone age, suggesting that GH might directly stimulate bone maturation.

3.5.6. Combined Pituitary Hormone Deficiency Patients with CPHD vary in their clinical findings because they have deficiencies of one or more of the other pituitary trophic hormones (ACTH, FSH, LH, PRL, or TSH) in addition to GHD (OMIM 262600). Although most cases are sporadic, familial forms of CPHD can have autosomal recessive, autosomal dominant, or X-linked modes of inheritance. The clinical features of familial CPHD are identical to those of cases of nongenetic etiology. The phenotype varies with the specific trophic hormone deficiencies, which occur in decreasing order: gonadotropins (FSH, LH) > ACTH > TSH. Associated gonadotropin deficiency causes sexual immaturity and primary ammenorrhea in females and small external genitalia and lack of beard growth in males. TSH deficiency might become severe after GH replacement and ACTH deficiency might contribute to recurrent hypo-glycemia. The severity of deficiencies of various trophic hormones exhibits interfamilial and intrafamilial variability in the various types of CPHD. Furthermore, the GH secretory responses to GHRH infusions vary from deficient to normal in different related individuals from the same families (55). HESX1 Mutations HESX1 is a transcription factor expressed in the thickened layer of oral ectoderm that gives rise to Rathke's pouch, the primordium of the anterior pituitary. Downregulation of HESX1 coincides with the differentiation of pituitary specific cell types. Dattani et al. found a missense HESX1 mutation (ARG53CYS) in a homozygous state in a brother and sister with septo-optic dysplasia, agenesis of the corpus callosum, and CPHD (OMIM 182230) (56). LHX3 Mutations Murine LHX3 mRNA accumulates in Rathke's pouch and is thought to be involved in differentiation of pituitary cells. Netchine et al. identified two families with CPHD (OMIM 262600) caused by mutations in the LHX3 gene (57). The phenotype associated with these mutations included the following: (1) severe growth retardation, (2) complete deficiency of all but one of the anterior pituitary hormones (ACTH), (3) elevated and anteverted shoulders with a short neck associated with severe restriction of rotation of the cervical spine, and (4) an enlarged anterior pituitary. The authors concluded that LHX3 is required for the proper development of all anterior pituitary cell types except corticotropes and that the rigid cervical spine phenotype is consistent with a function of LHX3 in the proper development of extrapituitary structures as well. PIT1 Mutations Defects in the PIT1 gene cause familial CPHD with a different phenotype (OMIM 173110). PIT1 is an anterior pituitary-specific transcription factor, that regulates the expression of GH, PRL, and TSH. PIT1 is also required for pituitary cellular differentiation and function. PIT1 has functional domains that enable transactivation of other genes including GH, PRL, and TSH on binding to these genes. At least six different PIT1 mutations causing autosomal recessive and two others causing autosomal dominant CPHD have been found in humans in a subtype of pan-hypopituitary dwarfism associated with GH, PRL, and TSH deficiency (see OMIM 173110).

Mutations of either the PIT1 or PROP1 genes can cause CPHD in humans. Both of these genes are members of the POU (Pit-1, Oct-1, and Unc-86) homeodomain family of transcription factors and they each play an important role in pituitary development. Pit-1 gene defects were first identified in Snell and Jackson dwarf mice, which lack somatotropes, lactotropes, and thyrotropes and have severe deficiencies of GH, PRL, and TSH (58). The Pit-1 (or GHF-1) gene, whose product binds to and activates GH, PRL, and TSH promoters, was found to have a T > G substitution at codon 261, giving a TGG (tryptophan)

to TGT (cysteine) in the Snell mice. A gross rearrangement of the Pit-1 gene was seen in the Jackson dwarf mouse. The deficiencies seen in these mice differ from most humans with CPHD, who have GH, TSH, gonadotropin, and ACTH deficiencies but increased PRL. In examining a subset of patients with GH, PRL, and TSH deficiency, eight different PIT1 mutations have been found in humans. Autosomal Recessive PIT1 Mutations The first PIT1 mutation reported to cause CPHD was a T > C transition in codon 172, which changes a CGA (Arg) to TGA (Ter) (59). The second PIT1 mutation reported was an A > G transition in codon 143, changing a CGA (Arg) to CAA (Gln) (60). Both mutations occur in the POU-specific domain of PIT1 and are thought to affect binding of the PIT1 protein to DNA. The third and fourth PIT1 mutations were found in two Dutch families who had postnatal growth failure with complete deficiencies of GH. PRL and T4 levels fell after treatment with rhGH in one case and were low prior to treatment in the other (61,62). One Dutch family had affected sibs whose T4 levels were initially normal. These sibs were homozygous for a C > G transversion in codon 158, which encoded a GCA (Ala) to CCA (Pro) substitution. This Ala158Pro mutation interferes with formation of PIT1 homodimers and greatly reduces transcription activation. The second Dutch family had affected sibs whose initial T4 levels were low. These sibs were genetic compounds with one deleted and one Ala158Pro PIT1 allele. Interestingly, this combination of defects was associated with more severe hypothyroidism and small anterior pituitary glands (62). These cases emphasize the importance of determining T4 and PRL levels and TSH responses to TRH administration in those with CPHD. Because GH and TSH deficiency often occur together, failure of subjects to have PRL and TSH responses to TRH should raise the question of their having PIT1 gene defects. A fifth PIT1 mutation was identified in a Thai patient who was homozygous for a G to T transversion in codon 25 converting a GAA(Glu) to TAA (Ter) codon. This mutation resulted in complete loss of the POU homeodomain, which is necessary for DNA binding (63). The sixth PIT1 mutation was found in a consanguineous family of Tunisian decent. All of the affected sibs were found to be homozygous for a T > G transversion in codon 135, converting a TTT (Phe) to TGT (Cys) in the POU-specific domain of PIT1 (64). Autosomal Dominant PIT1 Mutations Two dominant negative PIT1 mutations have been reported. Although the mechanism of action is not completely understood, neither of these mutations appear to inhibit binding of the mutant PIT1 protein to its target DNA. The first mutation, located in the POU homeodomain, is a C > T transition in codon 271 converting a CGG (Arg) to TGG (Trp) (65). Three unrelated patients (two adults and one infant) have been reported to be heterozygous for this Arg271Trp mutation. Both adults had pituitary hypoplasia and the 2-mo-old had a normal pituitary by imaging. These findings suggest that PIT1 might be necessary for anterior pituitary survival and that the 2-mo-old will develop pituitary hypoplasia with age. The second dominant negative mutation was a C > T transition of the 24th codon converting a CCT (Pro) to CTT (Leu). This mutation resides within the major transactivating domain of PIT1, which is highly conserved in different species (60). PROP1 Mutations PROP1 or Prophet of PIT1 is a pituitary-specific homeodomain factor that is required for development of somatotropes, lactotropes, and thyrotropes of the anterior pituitary and for expression of PIT1. Multiple PROP1 gene mutations cause an autosomal recessive CPHD that has a different phenotype in humans (OMIM 601538). In addition to the deficiencies of GH, PRL, and TSH seen in those with PIT1 defects, subjects with PROP1 defects also have deficiencies of LH and FSH, which prevent the onset of spontaneous puberty. In some cases, ACTH deficiency develops in later life. The various PROP1 mutations include (1) a C > T transition in codon 120, which encoded a TGC (Arg) to CGC (Cys) substitution, (2) a T > A transversion that encodes a TTC (Phe) to ATC (Ile) substitution at codon 117, and (3) 2-bp AG deletion in codon 101 (101delAG) that causes a frameshift and results in a premature stop at codon 109. The resulting protein products from all three of these different PROP1 mutations have greatly reduced DNA binding and transactivation abilities (66). 101delAG is a recurring mutation that is estimated to occur in about 55% of familial and 12% of sporadic CPHD cases (67). A fourth PROP1 mutation is a 2-bp GA deletion in codon 51 (51delGA) (68). Like the 101delAG mutation, the 51delGA mutation causes a frameshift that results in a premature stop codon. This mutation was found in 12% of familial and 21% of sporadic CPHD cases. X-Linked CPHD Lagerstrom-Fermer et al. reported a family that included affected males suffering from variable degrees of CPHD (OMIM 312000) (69). Some affected males died during the first day of life and had postmortem findings of hypoadrenalism, presumed to be the result of CPHD. Others had variable combinations of hypothy-roidism, delayed pubertal development, and short stature because of GHD. All surviving patients exhibited mild to moderate mental retardation. They found linkage with markers in the Xq25-q26 region. Furthermore, they found an apparent extra copy of the marker DXS102 in affected males and heterozygous carrier females, suggesting that a segment including this marker was duplicated.

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