Expanding the Phenotypic Spectrum and Variability of Endocrine Abnormalities Associated With TUBB3 E410K Syndrome

Abstract

Context:

A heterozygous de novo c.1228G>A mutation (E410K) in the TUBB3 gene encoding the neuronal-specific β-tubulin isotype 3 (TUBB3) causes the TUBB3 E410K syndrome characterized by congenital fibrosis of the extraocular muscles (CFEOM), facial weakness, intellectual and social disabilities, and Kallmann syndrome (anosmia with hypogonadotropic hypogonadism). All TUBB3 E410K subjects reported to date are sporadic cases.

Objective:

This study aimed to report the clinical, genetic, and molecular features of a familial presentation of the TUBB3 E410K syndrome.

Design:

Case report of a mother and three affected children with clinical features of the TUBB3 E410K syndrome.

Setting:

Academic Medical Center.

Main Outcome Measures:

Genetic analysis of the TUBB3 gene and clinical evaluation of endocrine and nonendocrine phenotypes.

Results:

A de novo TUBB3 c.1228G>A mutation arose in a female proband who displayed CFEOM, facial weakness, intellectual and social disabilities, and anosmia. However, she underwent normal sexual development at puberty and had three spontaneous pregnancies with subsequent autosomal-dominant inheritance of the mutation by her three boys. All sons displayed nonendocrine features of the TUBB3 E410K syndrome similar to their mother but, in addition, had variable features suggestive of additional endocrine abnormalities.

Conclusions:

This first report of an autosomal-dominant inheritance of the TUBB3 c.1228G>A mutation in a family provides new insights into the spectrum and variability of endocrine phenotypes associated with the TUBB3 E410K syndrome. These observations emphasize the need for appropriate clinical evaluation and complicate genetic counseling of patients and families with this syndrome.

We previously reported that a subset of recurrent heterozygous TUBB3 missense mutations alter the function of the neuronal-specific β-tubulin isotype 3 (TUBB3), interfere with normal axon development and maintenance, and produce a rare form of congenital paralytic strabismus and ptosis called congenital fibrosis of the extraocular muscles (CFEOM), often with additional clinical findings (1, 2). Among these cases, we reported that the heterozygous c.1228G>A TUBB3 mutation (putative E410K amino acid substitution) causes a distinct syndromic presentation that included fully penetrant CFEOM, facial weakness, stereotyped midface hypoplasia, developmental delay, intellectual disabilities, later-onset peripheral neuropathy, and Kallmann syndrome (KS; defects in olfaction associated with hypogonadotropic hypogonadism due to GnRH deficiency) (1). In some cases, the TUBB3 E410K syndrome also caused vocal cord paralysis, tracheomalacia, and cyclic vomiting syndrome (1). Thus, the E410K mutation defined a new genetic etiology for KS presumably resulting from aberrant axonal guidance.

In the initial descriptions of the TUBB3 E410K syndrome, all eight affected individuals harbored a de novo heterozygous c.1228G>A TUBB3 mutation consistent with an autosomal-dominant basis for this syndrome. Moreover, all E410K subjects who had reached puberty displayed hypogonadotropic hypogonadism with anosmia (ie, KS), incomplete or absent sexual maturation and, in keeping with the infertility typically associated with KS, none had a history of spontaneous fertility (1). These observations suggested an exclusive de novo etiology for the TUBB3 E410K syndrome that was always associated with a fully penetrant reproductive phenotype. In this report, we report the first family in which the TUBB3 c.1228G>A mutation initially arose de novo in a proband who surprisingly had normal sexual development and adult reproductive function, resulting in an autosomal-dominant transmission of the mutation to her three sons, all of whom displayed the clinical features of the TUBB3 E410K syndrome.

Materials and Methods

A Caucasian family in whom the proband and her three children were diagnosed with Moebius Syndrome was consented into an ongoing research study at Boston Children's Hospital. Following informed consent, their medical histories and records were reviewed and clinical evaluations were performed at Boston Children's Hospital. The TUBB3 coding exons and intron-exon boundaries were sequenced in salivary DNA of the proband, her parents, and her three children as previously reported (1, 2). Maternity and paternity of the proband's parents were confirmed by the appropriate inheritance of ten informative polymorphic markers.

Results

Clinical evaluation

Proband (II:4; Figure 1A)

Figure 1.

The TUBB3 c.1228G<A mutation (p.E410K) arose de novo in II:4 and then was transmitted as an autosomal-dominant mutation to III:1, III:2, III:3. A, Schematic structure of the pedigree. Squares denote males, circles denote females, and filled symbols indicate family members affected clinically by the TUBB3 E410K syndrome. The black triangle denotes II:4 as the proband. The absence or presence of the c.1228G<A mutation is indicated by a letter G or A, respectively. B, Chromatograms from the proband's unaffected father (I:1, top) and unaffected mother (I:2, middle) reveal wild-type sequence, whereas the chromatogram from the affected proband (II:4, bottom) reveals a heterozygous TUBB3 c.1228G<A mutation, represented by the double peak and indicated by a red arrow. The corresponding normal and mutated amino acid residues are indicated with a bracket under the codon triplet.

The proband was born at 38 weeks' gestation via spontaneous vaginal delivery to a 36-year-old G6P2 mother and 42-year-old father. She was born with nonprogressive bilateral ptosis, paralytic strabismus, and facial weakness, and was diagnosed with Moebius syndrome soon after birth. Her eyes were fixed in a down and out position, with an inability to elevate or adduct either eye. She underwent strabismus surgery and was found to have marked restriction to passive vertical and horizontal movements. She had a reduced sense of smell, delayed motor development, learning disabilities, difficulties modulating her emotions, and attended a college for the visually impaired. As a teenager she developed bilaterally high arched feet, hammer toes, and mild distal lower extremity weakness. Her pubertal onset and progression were normal with menarche between the ages of 12–13. She has had three spontaneous and uncomplicated pregnancies by two partners. At 31 years of age, her head circumference was below the 0.4th and her height was at the 50th. She had bilateral ptosis and her eyes were fixed in an exotropic and hypotropic position bilaterally. She scored anosmic (3/40) on the University of Pennsylvania Smell Identification Test (UPSIT) (3). She had developed some numbness and paresthesias in her feet. On nerve conduction studies, she had electrophysiologic evidence for a moderately severe sensorimotor polyneuropathy, with more prominent sensory involvement, that was most consistent with an axonal or mixed axonal/demyelinating process. Attempts to perform needle electromyography and brain magnetic resonance imaging (MRI) failed secondary to patient anxiety. At the time of evaluation, her menstrual cycles were irregular with a cycle length varying between 30 and 50 days. Her endocrine biochemical testing revealed normal anterior pituitary function with a normal basal cortisol (361 nmol/L) and adequate response to short synacthen (ACTH) stimulation (cortisol increased to 633 nmol/L after 30 min), normal thyroid axis (free T₄, 14.9 mmol/L; TSH, 1.1 mU/L), and normal IGF-I (1.19 mU/L) and prolactin (224 mU/L) levels. Serum estradiol (90 pmol/L), LH (7.3 U/L), and FSH (9.5 U/L), levels were compatible with early follicular phase of her menstrual cycle. A pelvic ultrasound confirmed adult-sized uterus and normal ovaries.

Proband's children (III:1-III:3; Figure 1A)

All three boys had congenital nonprogressive bilateral ptosis, paralytic strabismus with marked exotropia, limited vertical and horizontal eye movements, chin-up head position, and facial weakness. They also had hypotonia and global developmental delay with head circumferences of 2% (III:1), 15% (III:2), and 50% (III:3), and all three failed unsedated MRI of the brain secondary to their behavior. They had no history of cyclical vomiting and their general and neurological exams were otherwise normal. Limited nerve conduction studies in subjects III:1 and III:3 revealed no definite abnormalities.

The eldest son, III:1, 10 years old, was born full term and was not reported to have microphallus or cryptorchidism at birth. He had feeding difficulties leading to the diagnosis and repair of pyloric stenosis at a month of age. He subsequently showed behavioral difficulties and reported a reduced sense of smell. He underwent an anterior pituitary workup at 8 years of age due to concerns regarding his growth (height and weight at the second centile), which revealed normal thyroid functions, normal prolactin, and normal IGF-I levels. Dynamic GH axis evaluation initially showed a suboptimal GH response to arginine-GH stimulation, but a subsequent glucagon stimulation test showed normal GH response. A short ACTH stimulation test was normal and a LHRH stimulation test at age 8 years showed a prepubertal response in keeping with his age.

The middle son, III:2, 9 years old, was born at 37 weeks' gestation by emergency C-section for maternal pre-eclampsia and remained hospitalized for a week secondary to feeding difficulties. He had mild laryngomalacia and genital examination revealed an empty scrotum at birth that did not require therapy as subsequent examination revealed a high retractile testes. He developed clinical features consistent with autism spectrum disorder. Endocrine testing showed normal thyroid function, normal cortisol response to short ACTH stimulation, but low IGF-I levels. He subsequently had two arginine-GH stimulation tests, which revealed a suboptimal GH response (3.7 ug/dL and 5.73 ug/dL), and a repeat evaluation is pending. An LHRH stimulation test showed a prepubertal response in keeping with his age. He was not able to cooperate to ascertain his sense of smell.

The youngest son, III:3, 3 years old, was born full term via C-section because of increased maternal blood pressure and decreased fetal movements. Microphallus and bilateral cryptorchidism were noted at birth. He was lethargic during his first week of life and had two episodes of neonatal hypoglycemia, requiring transfer to the neonatal intensive care unit and initiation of treatment with steroids and thyroid hormone replacement for presumed hypopituitarism. He had no further hypoglycemic episodes. Neonatal brain MRI with pituitary imaging was read as normal. He subsequently has had two surgical procedures for correction of his undescended testes. He was too young to ascertain his sense of smell.

Proband's parents (I:1, I:2; Figure 1A)

The proband's mother (I:2) had three spontaneous abortions and two other children, both of whom had spina bifida. She reported normal timing and progression of puberty with menarche around age 12 years. Her UPSIT score was 30/40 at age 66 years (moderate hyposmia by UPSIT rating but at a normal 19th centile for age). The proband's father (I:1) reported normal timing of puberty on par with his peers and scored 35/40 on the Philadelphia Smell test at age 72 years (normosmia by UPSIT rating and 77th centile for age). There is no other family history of strabismus, neurologic problems, developmental delay, or reproductive difficulties.

TUBB3 sequencing results

The proband harbors a heterozygous de novo TUBB3 c.1228G>A mutation (TUBB3 E410K) that is absent in both of her biological parents and is transmitted on to all three of her affected children (Figure 1B).

Discussion

This case report reveals several new insights into the TUBB3 E410K syndrome. First, in contrast with the previous de novo TUBB3 c.1228G>A mutations that were associated with KS and consequent failure of sexual maturation, in this family the proband had full penetrance of CFEOM, facial weakness, developmental delay, and anosmia, but normal timing and progression of puberty along with normal adult reproductive function. Other anterior pituitary hormone function was also normal. She had three spontaneous pregnancies, each of which resulted in autosomal-dominant transmission of the TUBB3 c.1228G>A mutation to her three sons. Neonatal hypogonadism was present in two of her children (III:2, III:3) as evidenced by microphallus and/or cryptorchidism at birth suggesting that, in contrast with their mother, they may have hypogonadotropism due to GnRH deficiency that is likely to result in pubertal failure during adolescence. Although one child (III:1) did not have evidence of neonatal hypogonadism noted, his anosmia is suggestive that he too may have KS and may fail to go through puberty. Thus, this report documents the first inherited autosomal-dominant presentation of the TUBB3 E410K syndrome. Moreover, it establishes that this mutation can result in variable penetrance of hypogonadotropic hypogonadism despite the presence of anosmia, suggesting that TUBB3 mutations may have dissociated effects on the ontogeny of GnRH neurons and olfactory axons.

The lack of penetrance of the reproductive phenotype (ie, absence of GnRH deficiency) despite the presence of anosmia is intriguing because GnRH neurons are dependent on intact olfactory structures for their developmental migration into the hypothalamus (4). Incomplete penetrance of both reproductive and olfactory phenotypes or, in some cases, discordant penetrance of reproductive or olfactory phenotypes has been previously reported both within and across families with identical mutations in other KS-associated genes (FGF8, FGFR1, PROK2 and PROKR2) (5–7). Although oligogenicity (ie, co-occurrence of two independent KS-related genes) has been shown to explain this variable penetrance/expressivity in 10–15% of these KS patients (8), the oligogenicity hypothesis does not need to be implicated in patients harboring the c.1228G>A TUBB3 mutation; we previously reported that all pubertal-age subjects with this de novo mutation exhibited fully penetrant KS phenotype, suggesting that the mutation is solely sufficient for the expression of a full KS phenotype. Therefore, an alternative possibility is the presence of a genetic or epigenetic modifier in the proband that underlies the nonpenetrance of the reproductive phenotype. Finally, approximately 10% of KS patients with documented pathogenic mutations have been shown to spontaneously reverse their hypogonadism later in life, hinting at an exquisite plasticity of the hypothalamic GnRH neuronal network subject to stochastic or epigenetic modifiers (9, 10). Thus, a spontaneously reversible form of hypogonadotropism in the proband may also explain the spontaneous fertility and normal reproductive function seen in this subject.

Unlike the previously reported de novo E410K subjects, at least two children in this family had features suggestive of additional endocrine abnormalities including GH deficiency (suboptimal arginine-GH stimulation tests in oldest and middle sons) and history suggestive of hypothyroidism and/or hypoadrenalism in early neonatal life in the youngest child. Interestingly, mutations in a subset of other KS-associated genes have also been identified in patients with other pituitary hormone dysfunction (11, 12). However, biochemical testing confirming the hypothyroidism and hypoadrenalism in the youngest child was not available and hence studies of additional patients will be necessary to determine whether select TUBB3 mutations alter the ontogeny of additional hypothalamic-pituitary pathways beyond the hypothalmo-pituitary-gonadal axes.

In summary, we describe the first familial autosomal-dominant inheritance of the TUBB3 E410K syndrome and provide new insights into the spectrum and variability of endocrine phenotypes associated with this condition. These observations emphasize the need for appropriate clinical genetic counseling and followup of patients and families with the TUBB3 E410K syndrome.