Late infantile Hirschsprung disease–mental retardation syndrome with a 3-bp deletion in ZFHX1B

Case report.

A 48-year-old woman of normal birth from nonconsanguineous parents showed normal motor milestones until age 5. When the patient entered elementary school, she was noted as having mental retardation but received a full education in elementary school. She had no epileptic seizures. She sometimes had constipation although she did not need medication. At age 48, she presented with severe constipation, and was diagnosed with megacolon in a district hospital.

She was transferred to Fujita Neurologic Hospital and Fukui Medical University Hospital for further examination. She was 152 cm tall (mean ± 2 SD in the Japanese population, 155 ± 14 cm) and weighed 35 kg (mean ± 2 SD, 55 ± 10 kg). Her head circumference was 53 cm (mean ± 2 SD, 55 ± 3 cm), inner canthal distance was 35 mm (mean ± 2 SD, 33 ± 5 mm; range, 28 to 38 mm), interpupillary distance was 64 mm (mean ± 2 SD, 61 ± 4 mm; range, 56 to 65 mm), and palpebral fissure length was 32 mm (mean ± 2 SD, 30 ± 4 mm; range, 28 to 35 mm). She had neither microcephaly nor hypertelorism (figure 1A). Her feet showed mild pes cavus. Her abdomen was swollen with slightly decreased bowel movement. Although IQ evaluation was difficult, her word comprehension was almost normal, and she was able to speak a limited number of meaningful words or short sentences. She showed mild spastic paresis with hyperreflexia in the extremities. Results of the other neurologic examinations were normal.

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Figure 1. Facial appearance, megacolon, and colon mucosal pathology. (A) Photograph of the patient’s face. (B) Abdominal X-ray of the patient. The patient’s facial appearance is normal. Neither microcephaly nor hypertelorism is observed. The whole colon is markedly enlarged. (C) Immunohistochemical staining for S-100 (alpha and beta) in the muscular mucosae and submucosa. (D) Phosphorylated neurofilaments in the muscular mucosae and submucosa. (E) Hematoxylin and eosin staining of Meissner plexuses (arrow). (F) Microtubule-associated protein-2 immunohistochemical staining of Meissner plexuses (arrow). Bars indicate 20 μm.

Results of routine laboratory examinations were normal except for mild iron-deficient anemia. The Wassermann reaction was negative and thyroid functions were normal. Chromosomal analysis (G-band method) showed a normal karyotype of 46,XX without any chromosomal rearrangement. Brain MRI showed mild atrophy in the temporal lobes and no signs of agenesis of corpus callosum. EEG demonstrated mild slow background activities without any paroxysmal discharge. Abdominal X-ray demonstrated a marked enlargement of the colon (figure 1B). Echocardiogram showed no evidence of congenital heart diseases.

Colon-endoscopic examination and barium enema revealed narrowing in the distal segment of the colon without any organic lesion. Mucosal biopsy specimens from the narrowed portions revealed a mildly increased number of S-100 (alpha and beta) positive peripheral nerve bundles in the muscular mucosae and submucosa (figure 1C). These bundles contained axons as evidenced by immunopositivity for phosphorylated neurofilaments (figure 1D). There were also several Meissner plexuses containing a cluster of mature ganglion cells of which a few were immunopositive for microtubule-associated protein-2 (figure 1, E and F). These findings are not consistent with definite HSCR.

Methods.

We searched for a mutation of the ZFHXIB gene in the patient’s DNA. After written informed consent was obtained, genomic DNA was isolated from peripheral blood. The regions encompassing each exon and exon/intron boundary of the ZFHX1B gene were amplified by PCR, and sequenced directly with Sequenase version 2.0 (USB Co., Cleveland, OH). The region encompassing the mutation was subcloned into a pGEM–T Easy vector (Promega Co., Madison, WI), and the cloned plasmids containing normal and mutated regions were sequenced. To screen for the mutation in normal individuals, the region spanning the mutation was amplified with a forward primer 5′-CGGTATTGCCAACCCTCTG-3′ and a mismatched reverse primer 5′-ATCTACAGAGGCTTGT-AGAAGCTCG-3′(a mismatched site, T to G is italicized), which creates a restriction site for MwoI (restriction site: GCNNNNN(ΔAAC)NNGC). The amplified fragment (167 bp) containing the 3-bp deletion is cleaved into 142 bp and 25 bp fragments whereas the normal fragment (170 bp) is not cleaved.

Results.

A 3-bp deletion, AAC at the nucleotide position (np) 295–297, was identified in one allele of the gene (figure 2A). Sequencing of the cloned plasmid containing the mutated region clearly showed 3-bp deletion (see figure 2A). This mutation eliminates the amino acid Asn at codon 99 in exon 3 of the gene. Sequencing of all the exons and exon/intron boundaries revealed no other sequence alteration. The mutation was not found in 150 healthy control subjects in the restriction analysis of the 3-bp deletion in ZFHX1B gene (figure 2B).

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Figure 2. Heterozygous mutation in the ZFHX1B gene. (A) Left panel, direct sequencing of a PCR product spanning the mutation in the ZFHX1B gene. Middle panel, the sequence in a cloned plasmid containing the control region. Right panel, the sequence in a cloned plasmid containing the mutated region. Asterisks in the control sequence indicate the deleted nucleotides (AAC) at the nucleotide position 295–297 in the mutated region. The location of the 3–base pair (bp) deletion is shown as an arrow in the mutated sequence. (B) Restriction analysis of the 3-bp deletion in the ZFHX1B gene. MW = molecular weight marker; C1–15 = controls; P = patient.

Discussion.

We describe a 48-year-old woman with a late infantile form of ZFHX1B deficiency. Mutations in the ZFHX1B gene have been identified in HSCR–mental retardation syndrome.4-7⇓⇓⇓ This patient was noted as having mental retardation when she entered elementary school around age 5, whereas all other patients reported previously with ZFHX1B deficiency showed initial clinical symptoms in infancy (soon after birth or around age 1).4-7⇓⇓⇓ She had megacolon, but was not diagnosed with definite HSCR by rectal biopsy. Although all other reported patients had profound mental retardation, some patients did not have HSCR.6,7⇓ In addition, she had no characteristic features of facial dysmorphism, microcephaly, epilepsy, agenesis of corpus callosum, or congenital heart defects observed in other reported patients with ZFHX1B deficiency (see figure 1A).

The gene analysis newly identified a 3-bp deletion, AAC at the np 295–297, in one allele of the ZFHX1B gene (see figure 2). All mutations identified in previously reported patients are nonsense or frameshift mutations, presumably leading to nonfunctional ZFHX1B gene products.4-7⇓⇓⇓ This 3-bp deletion, non-frameshift mutation in exon 3 of this patient was subtle compared with the fatal mutations reported previously. We and Cacheux et al. reported that the ZFHX1B gene product (SIP1) was expressed ubiquitously and predominantly in fetal and adult human tissues.5,6⇓ Thus, SIP1 is thought to play a crucial role in normal brain development, although the molecular mechanism of SIP1 in neurogenesis is unclear. SIP1-knockout mice are useful for further understanding the mechanism of this protein.8 Other reports identified functional domains including zinc finger domains in the SIP1.9,10⇓ The identified 3-bp deletion in this patient is not located in important regions for SIP1 function, thought to be the Smad-binding domain or two-handed zinc finger domains. One plausible explanation for the mildness of the clinical features of this patient is that the non-frameshift 3-bp deletion affects SIP1 functions less severely than the other reported nonsense or frameshift mutations.

Moreover, this report suggests that screening ZFHX1B mutations in patients with the HSCR-mental retardation syndrome is warranted even in the absence of HSCR, facial dysmorphism, microcephaly, epilepsy, agenesis of corpus callosum, or congenital cardiac defects.