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. 2022;29(2):271-294.
doi: 10.15388/Amed.2022.29.2.13. Epub 2022 Jun 29.

Immunohistochemical Evaluation of BARX1, DLX4, FOXE1, HOXB3, and MSX2 in Nonsyndromic Cleft Affected Tissue

Mārtiņš Vaivads  1 , Ilze Akota  2   3 , Māra Pilmane  1
Affiliations

Immunohistochemical Evaluation of BARX1, DLX4, FOXE1, HOXB3, and MSX2 in Nonsyndromic Cleft Affected Tissue

Mārtiņš Vaivads et al. Acta Med Litu. 2022.

Abstract
in English, Lithuanian

Background: Nonsyndromic craniofacial clefts are relatively common congenital malformations which could create a significant negative effect on the health status and life quality of affected individuals within the pediatric population. Multiple cleft candidate genes and their coded proteins have been described with their possible involvement during cleft formation. Some of these proteins like Homeobox Protein BarH-like 1 (BARX1), Distal-Less Homeobox 4 (DLX4), Forkhead Box E1 (FOXE1), Homeobox Protein Hox-B3 (HOXB3), and Muscle Segment Homeobox 2 (MSX2) have been associated with the formation of craniofacial clefts. Understanding the pathogenetic mechanisms of nonsyndromic craniofacial cleft formation could provide a better knowledge in cleft management and could be a possible basis for development and improvement of cleft treatment options. This study investigates the presence of BARX1, DLX4, FOXE1, HOXB3, and MSX2 positive cells by using immunohistochemistry in different types of cleft-affected tissue while determining their possible connection with cleft pathogenesis process.

Materials and methods: Craniofacial cleft tissue material was obtained during cleft-correcting surgery from patients with nonsyndromic craniofacial cleft diagnosis. Tissue material was gathered from patients who had unilateral cleft lip (n=36), bilateral cleft lip (n=13), and cleft palate (n=26). Control group (n=7) tissue material was received from individuals without any craniofacial clefts. The number of factor positive cells in the control group and patient group tissue was evaluated by using the semiquantitative counting method. Data was evaluated with the use of nonparametric statistical methods.

Results: Statistically significant differences were identified between the number of BARX1, FOXE1, HOXB3, and MSX2-containing cells in controls and cleft patient groups but no statistically significant difference was found for DLX4. Statistically significant correlations between the evaluated factors were also notified in cleft patient groups.

Conclusions: HOXB3 could be more associated with morphopathogenesis of unilateral cleft lip during postnatal course of the disorder. FOXE1 and BARX1 could be involved with both unilateral and bilateral cleft lip morphopathogenesis. The persistence of MSX2 in all evaluated cleft types could indicate its possible interaction within multiple cleft types. DLX4 most likely is not involved with postnatal cleft morphopathogenesis process.

Kontekstas: Nesindrominis veido ir kaukolės nesuaugimas yra santykinai dažna įgimta anomalija, galinti turėti reikšmingų neigiamų pasekmių pediatrinės populiacijos asmenų sveikatos būklei ir gyvenimo kokybei. Aprašyta daugelis genų, kurie potencialiai gali lemti nesuaugimus, ir jų koduojami baltymai, aptariamas galimas jų dalyvavimas susidarant nesuaugimui. Dalis šių baltymų – tokie kaip homeozinis baltymas BarH-tipas 1 (BARX1), distalinis homeozinis genas 4 (DLX4), FOX E1 genas (FOXE1), homeozinis baltymas Hox-B3 (HOXB3) bei raumenų segmento homeozinis 2 genas (MSX2) – buvo siejami su kaukolės ir veido nesuaugimais. Patogeninio nesindrominio kaukolės ir veido nesuaugimo suvokimas galėtų suteikti daugiau žinių, kaip vertinti su nesuaugimais susijusius atvejus bei galimai taptų pradžios tašku, iš kurio kiltų naujos ar tobulesnės nesuaugimo gydymo alternatyvos. Šiame tyrime nagrinėjama, ar yra BARX1, DLX4, FOXE1, HOXB3 ir MSX2 genų turinčių ląstelių, pasitelkiant imunohistochemiją ir nagrinėjant įvairius nesuaugimo paveiktų audinių tipus, siekiant nustatyti galimą jų ryšį su nesuaugimo patogeneze.

Medžiagos ir metodai: Kaukolės ir veido nesuaugimo audinio medžiaga buvo įgyta nesuaugimo korekcinės operacijos metu iš pacientų, kuriems buvo nustatyta nesindrominio kaukolės ir veido nesuaugimo diagnozė. Audinio medžiaga buvo surinkta iš pacientų, kuriems yra vienpusis (n = 36) bei dvipusis lūpos nesuaugimas (n = 13) bei nesuaugęs gomurys (n = 26). Kontrolinės grupės (n = 7) audinio medžiaga gauta iš asmenų, neturinčių jokių kaukolės ar veido nesuaugimų. Teigiamo tyrimo faktoriaus ląstelių skaičius kontrolinės grupės ir pacientų grupės audiniuose buvo vertinamas pusiau kiekybiniu skaičiavimo metodu. Duomenys vertinti pasitelkus neparametrinius statistinius metodus.

Rezultatai: Buvo nustatyta statistiškai reikšmingų skirtumų tarp ląstelių, turinčių BARX1, FOXE1, HOXB3 bei MSX2, skaičiaus kontrolinėje grupėje bei pacientų, kurių audinys nesuaugęs, grupėse, tačiau nebuvo nustatyta jokių statistiškai reikšmingų skirtumų dėl DLX4. Statistiškai reikšmingos vertinamų faktorių koreliacijos buvo nustatytos ir pacientų, kurių audiniai nesuaugę, grupėse.

Išvados: HOXB3 galėtų būti labiau siejamas su vienpusio lūpos nesuaugimo morfopatogeneze po gimimo. FOXE1 ir BARX1 galima raiška tiek vienpusio, tiek ir abipusio lūpos nesuaugimo morfopatogenezėje. Kadangi MSX2 sistemingai yra visų nagrinėjamų nesuaugimo tipų atveju, galima daryti išvadą, kad MSX2 galimai yra susijęs su daugeliu nesuaugimo tipų. DLX4 veikiausiai nėra susijęs su nesuaugimo morfopatogenezės po gimimo procesu.

Keywords: cleft candidate genes; cleft lip; cleft palate; homeobox genes.

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Figures

Figure 1.
Figure 1.. BARX1 immunopositive cells within the control group and cleft affected tissues. (1A) Control tissue lacks BARX1-containing epitheliocytes and shows only a rare occurrence of BARX1 immunopositive connective tissue cells, BARX1 IMH (arrows), 200x. (1B) Unilateral cleft lip patient with an absence of BARX1 containing epitheliocytes and showing a few BARX1 immunopositive connective tissue cells (arrows), BARX1 IMH, 200x. (1C) Bilateral cleft lip patient absent of BARX1-containing epitheliocytes and demonstrating only a few BARX1 immunopositive connective tissue cells (arrows), BARX1 IMH, 200x. (1D) Cleft palate patient absent of BARX1-containing epitheliocytes and showing a rare occurrence of BARX1 positive connective tissue cells (arrows), BARX1 immunohistochemistry (IMH), 200x.
Figure 2.
Figure 2.. DLX4 immunopositive cells within the control group and cleft affected tissues. (2A) Control group with moderate to numerous DLX4-containing epitheliocytes and connective tissue cells, DLX4 IMH, 200x. (2B) Unilateral cleft lip patient with a few DLX4-containing epitheliocytes and connective tissue cells (arrows), DLX4 IMH, 200x. (2C) Bilateral cleft lip patient with few to moderate DLX4-containing epitheliocytes and connective tissue cells (arrows), DLX4 IMH, 200x. (2D) Cleft palate patient with a moderate number of weakly stained DLX4-containing epitheliocytes and a few DLX4 positive connective tissue cells (arrows), DLX4 IMH, 200x.
Figure 3.
Figure 3.. FOXE1 immunopositive cells within the control group and cleft affected tissues. (3A) Control with moderate to numerous FOXE1-containing epitheliocytes and few to moderate FOXE1-containing connective tissue cells, FOXE1 IMH, 200x. (3B) Unilateral cleft lip patient with a moderate number of FOXE1-containing epitheliocytes and the connective tissue cells, FOXE1 IMH, 200x. (3C) Bilateral cleft lip patient with a moderate number of FOXE1-containing epitheliocytes and a few FOXE1 positive connective tissue cells (arrows), FOXE1 IMH, 200x. (3D) Cleft palate patient with numerous FOXE1-containing epitheliocytes and few to moderate number of FOXE1 positive connective tissue cells, FOXE1 IMH, 200x.
Figure 4.
Figure 4.. HOXB3 immunopositive cells within the control group and cleft affected tissues. (3A) Control group with moderate HOXB3-containing epitheliocytes and few positive connective tissue cells (arrows), HOXB3 IMH, 200x. (3B) Unilateral cleft lip patient with a moderate number of HOXB3-containing epitheliocytes and the connective tissue cells, HOXB3 IMH, 200x. (3C) Bilateral cleft lip patient with moderate to numerous weakly stained HOXB3 epitheliocytes and a few positive connective tissue cells (arrows), HOXB3 IMH, 200x. (3D) Cleft palate patient with a moderate number of HOXB3-containing epitheliocytes and few to moderate HOXB3 positive connective tissue cells (arrows), HOXB3 IMH, 200x.
Figure 5.
Figure 5.. MSX2 immunopositive cells within the control group and cleft affected tissues. (3A) Control with a rare occurrence of MSX2-containing epitheliocytes (arrows) and absence of factor positive connective tissue cells, MSX2 IHC, 200x. (3B) Unilateral cleft lip patient with a moderate number of MSX2-containing epitheliocytes and a few positive cells in the connective tissue (arrows), MSX2 IHC, 200x. (3C) Bilateral cleft lip patient with a few MSX2-containing epitheliocytes (arrows) and a rare positive connective tissue cells (arrows), MSX2 IHC, 200x. (3D) Cleft palate patient with a moderate number of MSX2-containing epitheliocytes and with a few positive connective tissue cells (arrows), MSX2 IHC, 200x.
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