Novel Mutations for Nonsyndromic Cleft Lip and Palate in Two Consanguineous Palestinian Families

Abstract

Cleft lip and\or palate (CL/ CP) is the most common congenital malformation that affects the upper lip and the roof of the mouth. It is one of the most frequent congenital anomalies, affecting 1 in every 500 to 1000 births worldwide. In the Middle East, the incidence has variably been reported as 0.3 to 2.19 per 1000 lives. Higher incidence can be attributed to the high percentage of consanguineous marriages and low socioeconomic status (SES). Individuals with CL/ CP may face many problems in breathing, feeding, hearing, language and social integration. They need to undergo many reparative surgeries as well as other non-surgical therapies. CL/ CP may manifest in different forms including: cleft lip, cleft palate only (CPO) and cleft lip with palate (CLP) according to the anatomical structure. The etiology is multifactorial, multiple genes and environmental factors play a central role in the generation of the CL/ CP phenotype. In this study, next generation exome sequencing was performed to identify the mutations for three consanguineous Palestinian families collected from Ramallah and Hebron. Genotype to phenotype segregation within the families was validated by Sanger sequencing method. Carrier frequency within the healthy population was also determined in at least 200 healthy individuals. The exome sequencing revealed in CP-AL family a substitution mutation (BOD1_R112X, chr5: 173040162 G>A) in the second exon of the BOD1 gene, which converts the Arginine codon (CGA) to a stop codon (TGA). CP-BM family has a substitution mutation (IRF6_R250X, chr1:209964152 G>A). in exon 7 of the IRF6 gene, which also converts the Arginine codon (CGA) to a stop codon. Insertion mutation (CCDC141_I295L, chr2:179809274 ins A) in exon 6 of the CCDC141 gene has been found in CP-E family which leads to an early stop codon. Those mutations cause premature termination of transcription and release of incomplete, nonfunctional protein molecules. Validation by Sanger sequencing indicates that the IRF6 mutation is de-novo and there is no segregation for this mutation through the family. It also indicates that the ii BOD1 and CCDC141 mutations segregate perfectly with the phenotypes in the CPAL and CP-E families respectively under an autosomal recessive mode of inheritance. Further ascertainment of unaffected individuals from family CP-AL revealed that this mutation can't be the causative one for the clefting phenotype in this family rather it is causing another phenotype in the family. Using 200 Palestinian healthy controls, we could not find any of those three different mutations either in homozygous or heterozygous forms. Our study revealed that the identified mutations in the Palestinian CL/ CP patients are novel and occurred in two different gens, with zero carrier frequency in 200 healthy people. Keywords Cleft lip with or without cleft palate, Non Syndromic Cleft Palate, IRF6, CCDC141, BOD1.