How to Cite this Article: Sensi A, Ceruti S, Trevisi P, Gualandi F, Busi M, Donati I, Neri M, Ferlini A, Martini A. 2011. LAMM syndrome with middle ear dysplasia associated with compound heterozygosity for FGF3 mutations. Am J Med Genet Part A 155:1096–1101.
More than 400 syndromes with hearing impairment have been described and more than 100 show the involvement of the external ear [Reardon et al., 2004]. Recently a new, rare, autosomal recessive deafness syndrome has been described, with labyrinthine aplasia, microtia, and microdontia (LAMM) (OMIM #610706) [Tekin et al., 2007]. A first sporadic case of the syndrome was possibly reported by Hersh et al. , but it was only in 2007 that the syndrome was recognized and linked to mutations of the FGF3 gene [Tekin et al., 2007], already known to be involved in inner ear development [Represa et al., 1991; Mansour et al., 1993]. Up to now seven families (five Turkish, one Arabian, and one Somali), all consanguineous and with homoallelic mutations in affected subjects, have been described [Tekin et al., 2007, 2008; Alsmadi et al., 2009; Ramsebner et al., 2010]. Seven different mutations have been reported so far, four of them being represented by missense changes (p.L6P, p.G66C, p.S156P, p.R95W) and three by truncating mutations (p.R104X; c.254delT, c.617delG). The phenotype was invariant in the first six families, while in the most recent Somali family a variant LAMM phenotype, with variable inner and external ear abnormalities (but invariant dental findings), has been described.
Here we report on three patients from Europe (two sibs from Albania and one child from Italy) with clinical and radiological diagnosis of LAMM syndrome. Both families are non-consanguineous and show compound heterozygosity. Three novel FGF3 mutations, and recurrence of a previously reported nonsense mutation are described. Unlike the previously described cases, the two sibs in family A show hypoplasia/dysplasia of middle ear anatomical structures, therefore expanding the phenotypic spectrum of this disorder.
Two sibs, male and female, of non-consanguineous parents from Albania, both presenting with microtia, microdontia, and sensorineural hearing loss, were referred to us for audiologic and genetic assessment. Both the normal-hearing parents are in good health and a careful dysmorphological assessment failed to find any abnormality, with particular reference to external ears. The father did not show teeth abnormalities; the mother could not be examined because of a complete prosthesis for extensive caries (she denied teeth morphologic abnormalities or enlargement). The family history was negative for other cases of hearing loss, microtia, or teeth abnormalities. The couple's first daughter died from sudden infant death syndrome. At time of the examination the elder sib was a 12-year-old boy, born by normal delivery after uncomplicated full-term pregnancy. Profound sensorineural hearing loss was detected at the age of 6 months, when the parents realized that the baby did not respond to any sound. Pure tone audiometry showed profound loss, with only some perception for high volume low frequency sounds (threshold 80 dB at 125 Hz, 90 at 250 and 100 at 500). The boy started walking at the age of 18 months and is now attending a school for the hearing impaired, with good results.
The examination (Fig. 1) showed height, weight, span, and OFC within normal limits, occipital bone flattening, high forehead, very light eyebrows as well as light pigmentation of the skin, with small and scattered cafè au lait spots. Both ears were slightly low-set, small and abnormal: both were anteverted (the left one more hyperangulated) with a creased earlobe, and with bilateral absence of the superior crus of anthelix. Pits and tags were not observed, whereas lobulation of the left pinna was evident. Downslanting palpebral fissures, deepset eyes, high nasal bridge with hypoplastic alae nasi and small, widely spaced teeth were also noted (Fig. 1).
Renal ultrasound was normal and thyroid ultrasound did not show abnormalities. CT and MRI (Fig. 2) showed bilaterally labyrinthine aplasia and a very hypoplastic internal auditory canal; CT showed hypoplasia of the petrous pyramid (more evident on the right side) and, a bilateral hypo-dysplastic middle ear was observed (in particular hypoplasic incus long process and absent stapes as well as absence of oval and round windows).
The 9-year-old sister was born by normal delivery after uncomplicated full-term pregnancy. She has profound sensorineural hearing loss, detected by the age of 5 months, because of the familial history and microtia. Pure tone audiometry showed profound loss, with only some perception for high volume low frequency sounds (threshold 80 dB at 125 Hz, 100 at 250 and 110 at 500). The girl started walking at the age of 18 months and now is attending a school for the hearing impaired, with some learning difficulties.
The physical examination showed height, weight, span and OFC within normal limits, prominent forehead (similar to the mother), high nasal bridge, hypoplastic alae nasi, and small widely spaced teeth (Fig. 1). Both ears were slightly low-set, small, and anteverted (more symmetric than the brother's) with a creased earlobe and bilateral absence of the superior crus of anthelix (Fig. 1). Pits and tags were not observed; renal and thyroid ultrasounds were normal. CT and MRI of the petrous bones (Fig. 3) showed hypoplasia of petrous pyramids and bilateral labyrinthine and internal auditory canal aplasia; CT showed bilaterally hypo-dysplastic middle ear: in particular there were thick malleus handles and irregular malleus heads, hypoplasia of the incus long process, and absent oval and round windows. The stapes is present unlike in the sib.
The first child of a non-consanguineous couple from Italy was born at term of an uncomplicated pregnancy. The girl, 4 years, at the time of examination, started walking at the age of 18 months. ABR showed profound bilateral deafness. Both normal-hearing parents are in good health and a careful assessment failed to find any dental abnormality, but the mother had undergone two surgical procedures (early in childhood and at 14 years of age) for a unilateral external ear defect, described as similar to that of the child (no picture available). The family history was negative for other cases of hearing loss or external ear abnormalities. A previously obtained CT (only a written report was available) revealed normal middle ear and total labyrinthine aplasia bilaterally. Possible residual early otic vescicle remnant was observed ectopically in the left mastoid. The physical examination showed height, weight, span, and OFC within normal limits, slight hypoplastic alae nasi, downslanting palpebral fissures and small widely spaced teeth (Fig. 4). Both ears are slightly low set, small, and anteverted, absent bilaterally the superior crus of anthelix (Fig. 4) and show no tags or pits.
MATERIAL AND METHODS
Sequencing of the FGF3 Gene
Genomic DNA was extracted by standard methods from the whole blood of propositi and parents, after signing informed consent.
PCR amplification and sequencing were performed for the coding exons and exons-introns boundaries of fibroblast growth factor 3 gene (FGF3, GenBank accession number NM_005247.2). Utilized PCR primers were previously reported by Tekin et al., except for exon 1 amplification for which we designed new PCR primers (Fw 5′AGCACCTCGCAGCTGTCC3′; Rw 5′ GAGGCAGACGGTCTTTTCC3′).
The amplification conditions were as follows: 94°C initial denaturation (5 min), 94°C denaturation (30 sec), 60°C (30 sec) annealing, 72°C (30 sec) extension for 30 cycles followed by 72°C (7 min) final extension. All PCR reactions were carried out in a reaction volume of 25 L containing 25 ng of DNA template and 2.5 U Taq polymerase (Invitrogen, Carlsbad, CA), 1× Buffer, 0.75 mM MgCl2, 200 µM dNTPs, 0.5–1 µM each primer. For amplification of exon 1, 5% of glycerol was added to the PCR mix. PCR products were purified and sequenced using a Big Dye terminator cycle sequencing kit and analyzed with ABI Prism 3130 (Perkin-Elmer Applied Biosystems Division, Foster City, CA). Exon 3 amplification product was cloned into pCR-II vector (Invitrogen) and sequenced with M13 and T7 primers using a Big Dye terminator cycle sequencing kit and analyzed with ABI Prism 3130 (Perkin-Elmer Applied Biosystems Division).
Sequence analysis in both affected sibs and parents from Family A led to the identification of a maternal A > G substitution within exon 2 of the FGF3 gene at nucleotide c.317, predicted to cause the missense change p.Y106C. This mutation was present in compound heterozygosity with a paternal mutation in exon 3 consisting of a 2-nucleotide deletion (c.457-458 del TG), leading to frameshift and predicted to introduce a premature termination codon (p.W153VfsX51). In Patient B the paternal missense mutation p.Y49C was found in compound heterozygosity with the maternal nonsense mutation (p.R104X). Analysis of 50 unrelated control subjects (100 chromosomes) of the same European background did not show any of the two newly reported missense variations.
LAMM (OMIM #610706) is a recently identified syndrome associated to homozygous mutations in the FGF3 gene [Tekin et al., 2007]. The phenotype of LAMM syndrome is characterized by external ear abnormalities, small widely spaced teeth and labyrinthine aplasia. A minor dysmorphologic sign that could be worth noticing is the hypoplasia of alae nasi, observed in both the reported patients from Family A (Fig. 1). The sign is mild in Patient B (Fig. 4), but it is recognizable in many of the reported pictures of the LAMM patients. A similar nose is found in the 22q11.2 deletion syndrome, a syndromic form of hearing loss with a variable phenotype, and showing dysmorphic pinna and occasionally labyrinthine abnormalities.
The involvement of the middle ear structures, previously excluded in this condition [Tekin et al., 2007, 2008; Alsmadi et al., 2009] is documented in both sibs from Family A. Middle ear defects could be coincidental or they may represent an inconstant, variable finding in LAMM syndrome. The association between complete labyrinthine aplasia and middle ear malformations has been recently reported by Ozgen et al.  in a series of nine cases (LAMM syndrome was not diagnosed in any of them): middle ear volumes were decreased in most of the ears; moreover stapes was aplastic in one ear and dysplastic in five. Moreover, the inactivaction of the genes EYA1 and SIX1, both involved in otic placode specification, causes branchio-otorenal syndrome, which is characterized by primary inner ear aplasia or severe cochlear malformations associated to outer and middle ear malformations. FGF3 too is involved in placode formation [Itoh and Ornitz, 2008]; thus it is conceivable that middle ear involvement could actually be associated with FGF3 inactivation and not merely a coincidental finding.
Tekin et al.  looked for FGF3 mutations in eight subjects with inner ear abnormalities, without LAMM syndrome, and did not find any mutation [Tekin et al., 2008]. However, Ramsebner et al.  found FGF3 mutations in a Somali family with a LAMM variant with different and variable inner ear abnormalities. In this regard, the finding of the otocyst remnant described in Patient B (similar to that found in one patient by Tekin et al. ) confirms some possible inner ear development in the LAMM phenotypic spectrum.
We add three novel mutations in FGF3 gene associated with the LAMM phenotype. The first mutation is a missense mutation in exon 2, predicted to change a polar neutral tyrosine residue with a non-polar neutral cysteine at the 106 position, in the final product of FGF3. The introduction of a cysteine residue in addition to native cysteine residues at position 50 and 115 may result in alteration of a secondary structure that results in loss of function. Moreover, the tyrosine position 106 of FGF3 is conserved in a variety of organisms from fishes to frogs to humans (Fig. 5), supporting a pathogenic role for its substitution. The second mutation, observed in compound heterozygosity with the first one in the two sibs of Family A, is a frameshift mutation due to the deletion of two nucleotides in exon 3 (p.W153VfsX51, c.457–458 del TG). Heparan binding sites of FGF3 have been located on the carboxyterminal portion of the peptide, after residue 160, so it seems likely that the mutation is amorphic or possibly hypomorphic. The third mutation here reported (Y49C) substitutes another conserved (Fig. 5) non-polar tyrosine residue with a polar cysteine, adjacent to the cysteine in position 50. Its pathogeneticity seems likely, also because the serine residue should be just in the specific receptor interaction site [Plotnikov et al., 1999].
A not yet resolved issue is represented by the relationship between FGF3 and another rare syndromic deafness: The otodental syndrome. This syndrome is an autosomal dominant disorder characterized by grossly enlarged molar teeth and progressive high frequency hearing loss of probable cochlear type and has been reported to be associated with FGF3 deletions [Gregory-Evans et al., 2007]. In particular, three overlapping heterozygous deletions were identified segregating with the otodental phenotype in three families and the minimal common overlapping region included only the FGF3 gene [Gregory-Evans et al., 2007]. However, it is not clear whether FGF3 haploinsufficiency can cause otodental syndrome, because the phenotype was not observed in the heterozygous parents of LAMM syndrome patients [Tekin et al., 2007, 2008; Alsmadi et al., 2009; Ramsebner et al., 2010]. We add to the previous reports, three further heterozygous carriers of novel FGF3 mutations without signs of otodental syndrome, supporting the alternative possible explanation put forward by Gregory Evans of a position effect of the deletion of FGF3 on the FGF4 and FGF19 neighboring genes [Gregory-Evans et al., 2007].
Finally, the finding of the external ear abnormality in the heterozygous mother of Patient B is intriguing. A coincidental finding cannot be excluded, but a microtia requiring two surgical procedures is not frequent in general population and could be related to her heterozygosity for p.R104X nonsense mutation in FGF3. She does not have hearing loss and most importantly, her teeth are normal. It seems unlikely that unilateral microtia in this case represents a minimal sign of otodental syndrome, especially considering that microtia is not reported in the otodental syndrome (Jorgenson et al.  just reported “protuberant ears”). Mehes found morphogenetic variants in parents of children with recessive malformation syndromes and hypothesized a somatic second hit mosaicism [Méhes, 1990]: it is tempting to speculate a similar mechanism in this case. Further analysis of FGF3 in different cochlear, vestibular, and dental abnormalities are required in order to better define the phenotypic spectrum associated with FGF3 mutations.
We would like to thank Dr. Eleonora Bentivogli for her linguistic support in preparing the text.