A clinical and multi­omics study of Van der Woude syndrome in three generations of a Chinese family.

Previous studies have suggested that pathogenic variants in interferon regulatoryse factor 6 (IRF6) can account for almost 70% of familial Van der Woude Syndrome (VWS) cases. However, gene modifiers that account for the phenotypic variability of IRF6 in the context of VWS remain poorly characterized. The aim of this study was to report a family with VWS with variable expressivity and to identify the genetic cause. A 4­month­old boy initially presented with cleft palate and bilateral lower lip pits. Examination of his family history identified similar, albeit milder, clinical features in another four family members, including bilateral lower lip pits and/or hypodontia. Peripheral blood samples of eight members in this three­generation family were subsequently collected, and whole­exome sequencing was performed to detect pathogenic variants. A heterozygous missense IRF6 variant with a c.1198C>T change in exon 9 (resulting in an R400W change at the amino acid level) was detected in five affected subjects, but not in the other three unaffected subjects. Moreover, subsequent structural analysis was indicative of damaged stability to the structure in the mutant IRF protein. Whole­transcriptome sequencing, expression analysis and Gene Ontology enrichment analysis were conducted on two groups of patients with phenotypic diversity from the same family. These analyses identified significant differentially expressed genes and enriched pathways in these two groups. Altogether, these findings provide insight into the mechanism underlying the variable expressivity of VWS.

Osteocalcin and regulatory cytokine imbalance in children with congenital cleft lip and palate.

The aim of the work was a comprehensive assessment of the cytokine system and peripheral blood osteocalcin with the establishment of features of their interconnections in children with congenital cleft lip and palate (CCLP) in comparison with corresponding controls at different age periods. Levels of IL17, IL4, IL6, IL1ß, IFNγ and osteocalcin were analyzed by enzyme immunoassay in the peripheral blood of 80 children (0-12 months, 1-3 years, 4-9 years, 10-15 years) with CCLP and age-appropriate control of healthy individuals (40 people). An analysis of the obtained data shows that in children with CCLP we revealed significant differences between pro-inflammatory (IL1ß, IL6, IL17), regulatory (IFNγ), anti-inflammatory (IL4) cytokines and osteocalcin compared with controls. Differences were found in the content of IL17, IFNγ, IL4 and osteocalcin in healthy children and in children with CCLP in postnatal ontogenesis. Cytokine deregulation of immunosteogenesis in CCLP, leading to a significant deficit of osteocalcin in the first year of life due to imbalance of the cytokine profile: discordant IL17, IFNγ and IL4 were detected. Obtained data are undoubtedly important in the future for developing new strategies for targeted therapy aimed at normalizing osteocalcin levels at different age periods in children with CCLP.

Targeted next­generation sequencing for research and diagnostics in congenital heart disease, and cleft lip and/or palate.

Congenital heart disease (CHD), and cleft lip and palate (CLP) are currently the most common types of structural malformation in infants. Various methods have been used to identify the disease­associated genes. However, targeted next­generation sequencing (NGS) is not yet considered an option for routine use. Thus, the present study aimed to assess the safety and feasibility of using targeted NGS in patients with CHD concomitant with CLP. Between November 2015 and May 2017, a total of 17 patients with CHD concomitant with CLP, who were excluded from a diagnosis of trisomy syndrome, were selected at The Second Xiangya Hospital of Central South University (Changsha, China). Genomic DNA was extracted from peripheral blood samples of the patients. The copy number variants (CNVs) were determined by conducting a single nucleotide polymorphism (SNP) array with Illumina HumanOmni1­Quad Beadchip, while information on other gene mutations was obtained from targeted sequencing. The functions of gene mutations were then predicted using the PolyPhen­2, SIFT and Mutation Taster tools. Finally, Sanger sequencing was used to verify the mutations. The results identified no pathogenic mutations in CNVs analyzed by high­throughput SNP sequencing. Targeted NGS results demonstrated that 10 patients (58.8%) carried gene mutations, including 4 (23.5%) genetically diagnosed cases and 6 (35.3%) cases with unknown etiology. The 4 known diseases were Opitz G/BBB syndrome caused by MID1 gene mutation, Loeys­Dietz syndrome caused by TGFBR1 gene mutation, Ritscher­Schinzel/3C syndrome caused by KIAA0196 gene mutation and CHARGE syndrome caused by CHD7 gene mutation. The remaining 6 cases were not genetically diagnosed, although they carried candidate genes. In conclusion, the present study demonstrated that targeted NGS was an effective and accurate candidate gene detection method in patients with CHD concomitant with CLP.

Evaluation of Oxygen Saturation and Heart Rate During Intraoral Impression Taking in Infants With Cleft Lip and Palate.

OBJECTIVE: The aim of the study was to evaluate the changes in oxygen saturation and heart pulse during intraoral impression taking from infants with cleft lip and palate (CLP) at onset of presurgical orthopedic therapy. SUBJECTS AND METHODS: In our study, 21 uni- and bilateral infants with CLP (9 female, 12 males, mean age 5.90 ±â€Š2.46 days) were monitored and heart pulse and oxygen saturation were measured under operating conditions before any intervention (T1), after delivery of 50% supplemental oxygen (T2), during impression taking with oxygen support (T3), and immediately before the discharge from the operating room (T4). RESULTS: Statistically significant differences were found in the average oxygen saturation levels (P < 0.01), but not in heart rates (P > 0.05) between T1, T2, T3, and T4. Decreases in the oxygen levels from T2 to T3 were noted (P < 0.01). Overall, oxygen levels increased from T1 to T4 (P < 0.01). CONCLUSIONS: Oxygen saturation levels may decrease during intraoral impression taking in infants with CLP despite the supplemental oxygen under operating conditions. No changes in the heart rate during the procedure showed that the cardiac output was sufficient, whereas the decrease in oxygen saturation demonstrated hypoventilation because of the anatomic structure and impression-taking process. It may be advised that the impression should be taken under the supervision of the anesthetist, with monitoring of, and supplying oxygen to, the infant with CLP.

Hematological parameters in patients of cleft lip and cleft palate with special reference to eosinophil counts.

INTRODUCTION: Birth abnormalities like cleft lip and cleft palate account for about 1.4 per 1000 live births in India. These are seen to be associated with a high incidence of eosinophilia which delays the surgical management of these patients. AIMS: The aim of this paper is to study the hematological parameters in patients of cleft lip and cleft palate. PATIENTS AND METHODS: A total of 223 cases of cleft lip and cleft palate were taken up for the study. Hematological parameters including hemoglobin, total leukocyte count, differential leukocyte count, absolute eosinophil count, and red cell indices were studied. RESULTS: Anemia was found in 182/223 (81.63%) cases which was most commonly of microcytic hypochromic type. Eosinophilia was seen in 46/223 (20.60%) cases. CONCLUSIONS: Many cleft lip and cleft palate patients show high eosinophil counts. Absolute eosinophil count was found to be a better parameter for assessment of eosinophils.

Lower concentrations of B-vitamin subgroups in the serum and amniotic fluid correlate to cleft lip and palate appearance in the offspring of A/WySn mice.

PURPOSE: The pathogenesis and prevention of cleft lip and palate (CL/P) have been studied mainly in clinical and animal experiments. A prophylactic poly-B-vitamin substitution during the first months of pregnancy has provided the most encouraging results for the prevention of CL/P recurrence in families at risk. In vitro studies of the palatal organ in an A/WySn mouse model have confirmed the positive influence of B-vitamins on palatal development. The present animal study was performed to analyze different B-vitamin concentrations in the serum and amniotic fluid of A/WySn mice according to the appearance of CL/P in their offspring. MATERIAL AND METHODS: Concentrations of different B-vitamins (B1, B2, B3, B5, B6, and folic acid) in serum and amniotic fluid were analyzed by high-performance liquid chromatographic detection. Immunohistochemical staining against thiamin-1 receptor was performed on histologic midface sections of A/WySn fetuses with (n = 12) and without (n = 14) CL/P. RESULTS: Vitamin B5 (P < .001) and folic acid (P < .004) concentrations in the amniotic fluid of dams with CL/P were significantly lower than in dams without CL/P. Serum concentrations of folic acid (P = .5) and B5 (P = .4) showed no difference between the 2 groups. Dams with CL/P had significantly lower thiamine concentrations in serum (P = .01) and amniotic fluid (P < .001). Histologic midface sections presented high thiamin-1 receptor expression in the palatal shelf of fetuses with CL/P. CONCLUSION: A decreased use or uptake of some B-vitamin subgroups (B1, B5, and folic acid) in amniotic fluid and serum (vitamin B1) was correlated to an increased cleft appearance in A/WySn mice. The high thiamin-1 receptor expression in the palatal tissue of mouse fetuses with CL/P may be caused by a decreased availability of vitamin B1.

Intraoperative vascular anatomy, arterial blood flow velocity, and microcirculation in unilateral and bilateral cleft lip repair.

BACKGROUND: Cleft lip repair aims to normalize the disturbed anatomy and function. The authors determined whether normalization of blood circulation is achieved. METHODS: The authors measured the microcirculatory flow, oxygen saturation, and hemoglobin level in the lip and nose of controls (n = 22) and in patients with unilateral and bilateral cleft lip-cleft palate. The authors measured these parameters before lip repair (n = 29 and n = 11, respectively), at the end of lip repair (n = 27 and 10, respectively), and in the late postoperative period (n = 33 and n = 20, respectively). The arterial flow velocity was measured in unilateral groups at the same time points (n = 13, n = 11, and n = 12, respectively). Statistical differences were determined using analysis of variance. RESULTS: Before surgery, the arterial flow velocities and microcirculation values were similar on each side of the face and between groups. The microcirculatory flow was significantly higher in the prolabium of bilateral patients than in the philtrum of controls. All circulation values in unilateral and bilateral patients in the late postoperative period were within the range of controls and of those before surgery. Intraoperatively, the authors consistently found a perforating artery on the superficial side of the transverse nasalis muscle. CONCLUSIONS: There appears to be no intrinsic circulatory deficit in unilateral and bilateral cleft lip-cleft palate patients. The increased flow in the prolabium indicates a strong hemodynamic need in this territory, compelling its vascular preservation. Whether surgical preservation of the nasalis perforator artery is of long-term benefit should be addressed in future studies. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Are routine pre-operative blood tests required in children undergoing primary cleft lip and/or palate repair?

Primary surgical repair of cleft lip and/or palate is performed before 9 months of age, often representing the first surgical intervention these children encounter. Obtaining pre-operative blood tests in young children often produces much anxiety for all involved. We reviewed the electronic data of 282 children over a five-year period undergoing primary cleft repairs to determine the value of pre-operative full blood count, and transfusion requirements. Of these, three children required post-operative blood transfusion. In two cases concurrent illness contributed to transfusion requirement. To determine if our findings were consistent with those at other Cleft Centres, the views of primary cleft surgeons in the UK and Ireland were obtained using a questionnaire. A 96% response rate was achieved. The majority of cleft surgeons stated they no longer request routine pre-operative blood tests. Few could recall any of their patients requiring transfusion, and in those that did there was an underlying medical condition contributing to transfusion requirement, and an equal number whom could not have been predicted pre-operatively. The benefit of obtaining routine full blood count and group and save in children undergoing cleft repair is small in comparison to the stress caused from obtaining these bloods, and has significant cost implications to the Health Service.

Relación entre los niveles de ácido fólico, vitamina B12 y homocisteína materna y los defectos de tubo neural y labio hendido / Relation between levels of folic acid, vitamin B12 and maternal homocysteine with neural tube defects and cleft lip

El objetivo del trabajo fue determinar la asociación entre los niveles de ácido fólico, vitamina B (Vit B12) y homocisteína (Hci) maternos, con defectos del tubo neural (DTN) y labio hendido (LH) con y sin paladar hendido (c/s PH). Se realizó un estudio tipo casos y controles. Casos, con diagnóstico de DTN y LH c/s PH (n=36) y cuatro controles hospitalarios por caso (n=141). Se incluyeron recién nacidos (RN) y lactantes hasta 12 meses de edad. Las variables de pareamiento fueron: edad del RN o lactante, etnia y hospital. Un 23 por ciento de etnia Tarahumara y 77 por ciento mestizos. Se determinó ácido fólico intraeritrocitario (AFI), plasmático (AFP) y Vit B12 por radioinmunoensayo, la Hci por inmunoensayo de fluorescencia polarizada. Se consideró deficiencia si el AFI fue <160 ng/mL, AFP <3.5 ng/ mL y la Vit B12 <200 pg/mL e hiperhomocisteinemia, si Hci >15 J.mol/L. El análisis estadístico se realizó a través de regresión logística condicionada. Se identificó deficiencia de AFI en el 22 por ciento de las mujeres cuyos recién nacidos o lactantes presentaron algún tipo de defecto congénito y en el 12 por ciento de los controles. La relación entre AFI y DTN, LH c/s PH ajustada por edad materna, exposición a plaguicidas y zona de residencia fue RM 2,96 (IC 95 por ciento 0,92-9,46). No se encontraron diferencias en los niveles de Hci ni de Vit B12. Conclusiones: Nuestros resultados sugieren que RN cuyas madres cursan con una deficiencia de AFI tienen mayor riesgo de presentar DTN y LH c/s PH.

Objective: To determine the association between maternal folate deficiency, neural tube defects (NTDs), and cleft lip, with and without cleft palate (CL/P). Material and methods: A case/control study was conducted. The cases included subjects with diagnoses of NTD and CL/P (n=36) and four hospital controls per case (n=141); the study included newborns (NBs) and nursing babies up to 12monthsof age. The parameter variables were the following: the age of the NB or nursing baby, the ethnic group, and the hospital of origin. The Tarahumara ethnic group made up 23 percent of the cases, while 77 percent were mestizos. The red cell folate (RCF), the plasma folie acid (PFA), and the vitamin B12 levels were determined by radioimmunoassay and the homocysteine levels by polarized fluorescence immunoassay. A deficiency was considered to be present if the RCF were <160 ng/mL, the PFA <3.5 ng/mL and the vitamin B <200 pg/ mL; hyperhomocysteinemia was defined as HC >15 J,mol/L. The statistical analysis was carried out through of conditional logistic regression. Results: An RCF deficiency was identified in 22 percent of the women whose newborn or nursing babies presented with some type of congenital defect and in 12 percent of the controls. The correlation adjusted by maternal age, exposure to pesticides and zone of residence was OR 2.96 (CI 95 percent 0.92-9.46). There was no difference in vitamin B12 or homocysteine levels between groups. Conclusions: Our results suggest that newborns whose mothers present with an RCF deficiency have an increased risk of displaying NTD and CL/P.

Association between school performance, breast milk intake and fatty acid profile of serum lipids in ten-year-old cleft children.

The fatty acid profiles of serum lipids were examined in 53 ten-year-old cleft children. The children presented with different cleft types (cleft lip, cleft lip and palate, isolated cleft palate and submucuous cleft palate) and were recruited from the Finnish Cleft Center. We also studied associations between serum lipid fatty acids and early breast milk intake, cognitive development in terms of preschool language learning and school achievement. The fatty acid profiles of serum lipids did not differ between boys and girls. The proportion of myristic acid in serum cholesteryl esters (CE) was higher and proportion of nervonic acid in phospholipids (PL) lower in children with isolated palatal clefts than in those with submucuous clefts. Out of the present children, 30% and 60% received breast milk less than 1 or 3 months, respectively. The proportions of docosahexaenoic acid in CE and in PL were significantly higher in the children whose breast milk intake was longer than 3 months. The number of children requiring special education was higher among those who received breast milk less than 1 month than among those with longer breast milk intake. In conclusion, the fatty acid profiles of serum lipids seem to be comparable among children with different cleft types. Short breast milk intake was associated with poorer school performance.

Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults.

BACKGROUND: Clefts of the lip, alveolus, and palate (CLPs) rank among the most frequent and significant congenital malformations. Leu10Pro and Arg25Pro polymorphisms in the precursor region and Thr263Ile polymorphism in the prodomain of the transforming growth factor beta1 (TGF-beta1) gene have proved to be crucial to predisposition of several disorders. METHODS: In this study, polymorphism analysis was performed by real-time polymerase chain reaction (LightCycler) and TGF-beta1 levels determined by enzyme-linked immunosorbent assay. RESULTS: Only 2/60 Caucasian non-syndromic patients with CLP (3.3%) carried the Arg25Pro and another 2/60 patients (3.3%) the Thr263Ile genotypes, whereas, in a control group of 60 healthy Caucasian blood donors, these heterozygous genotypes were more frequent 16.7% having Arg25Pro (10/60; p < 0.035) and 10,0% having Thr263Ile (6/60), respectively. TGF-beta1 levels in platelet-poor plasma of heterozygous Arg25Pro individuals were lower than those of homozygous members (Arg25Arg) in the latter group, but this discrepancy narrowly failed to be significant. Although polymorphisms in codon 10 and 25 were associated with each other, no difference was found between patients and controls concerning the Leu10Pro polymorphism. CONCLUSIONS: The genetic differences in codons 25 and 263 suggest that TGF-beta1 could play an important role in occurrence of CLP, however, functional experiments will be required to confirm the mechanisms of disturbed development.

The effect of follow-up on limiting non-participation bias in genetic epidemiologic investigations.

The use of a comprehensive follow-up strategy to limit non-participation bias was evaluated in a population-based case-control study of orofacial clefts. Birth parents were requested to provide exposure data, and index children and parents were asked to provide blood specimens. Follow-up included telephone or postal reminders every two weeks for up to three months. Consent to participate was received from 281 (76.6%) case mothers and 246 (72.4%) case fathers. The corresponding totals for controls were 279 (54.7%) and 245 (49.8%). Evaluation of participation rates by intensity of follow-up showed that 23% of case and 18% of control families consented without reminders (first stage); 81% of cases and 83% of controls agreed following one or two reminders (second stage); and the remainder of participants consented following three or more reminders (final stage). Cumulative distributions of sociodemographic characteristics differed little between second and final stage participants. Odds ratios for maternal multivitamin use were similar between second and final stage participants, whereas those for maternal and paternal smoking tended to decline. Although follow-up measures were necessary to enroll most families, use of more than two reminders did not appear to increase the representativeness of the sample; however, termination of recruitment after only two reminders would have led to different conclusions. Future studies require data collection protocols that encourage participation from all population subgroups, and one alternative is presented.