Impact of rare variants in ARHGAP29 to the etiology of oral clefts: role of loss-of-function vs missense variants.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent, complex congenital malformation. Genome-wide association studies (GWAS) on NSCL/P have consistently identified association for the 1p22 region, in which ARHGAP29 has emerged as the main candidate gene. ARHGAP29 re-sequencing studies in NSCL/P patients have identified rare variants; however, their clinical impact is still unclear. In this study we identified 10 rare variants in ARHGAP29, including five missense, one in-frame deletion, and four loss-of-function (LoF) variants, in a cohort of 188 familial NSCL/P cases. A significant mutational burden was found for LoF (Sequence Kernel Association Test, p = 0.0005) but not for missense variants in ARHGAP29, suggesting that only LoF variants contribute to the etiology of NSCL/P. Penetrance was estimated as 59%, indicating that heterozygous LoF variants in ARHGAP29 confer a moderate risk to NSCL/P. The GWAS hits in IRF6 (rs642961) and 1p22 (rs560426 and rs4147811) do not seem to contribute to the penetrance of the phenotype, based on co-segregation analysis. Our data show that rare variants leading to haploinsufficiency of ARHGAP29 represent an important etiological clefting mechanism, and genetic testing for this gene might be taken into consideration in genetic counseling of familial cases.

Protocol to cryopreserve and isolate nuclei from adipose tissue without dimethyl sulfoxide.

Cryopreservation injuries involve nuclear DNA damage. A protocol for cryopreserving and isolating adipocyte nuclei is proposed. Adipose tissue samples were directly analyzed (NoCRYO-0h), or stored at -196°C for 7 days without 10% dimethyl sulfoxide (DMSO) (CRYO-WO-DMSO) or with DMSO (CRYO-W-DMSO). To determine the effect of DMSO on cryopreservation treatment, adipose tissue samples were stored at 4°C for 24 h with 10% DMSO (NoCRYO-W-DMSO-24h) and without (NoCRYO-WO-DMSO-24h). Samples were processed in isolation buffer, and nuclear integrity was measured by flow cytometry. The coefficient of variation, forward scatter, side scatter, and number of nuclei analyzed were evaluated. Pea (Pisum sativum) was used to measure the amount of DNA. All groups contained similar amounts of DNA to previously reported values and a satisfactory number of nuclei were analyzed. CRYO-W-DMSO presented a higher coefficient of variation (3.19 ± 0.09) compared to NoCRYO-0h (1.85 ± 0.09) and CRYO-WO-DMSO (2.02 ± 0.02). The coefficient of variation was increased in NoCRYO-W-DMSO-24h (3.80 ± 0.01) compared to NoCRYO-WO-DMSO-24h (2.46 ± 0.03). These results relate DMSO presence to DNA damage independently of the cryopreservation process. CRYO-W-DMSO showed increased side scatter (93.46 ± 5.03) compared to NoCRYO-0h (41.13 ± 3.19) and CRYO-WO-DMSO (48.01 ± 2.28), indicating that cryopreservation with DMSO caused chromatin condensation and/or nuclear fragmentation. CRYO-W-DMSO and CRYO-WO-DMSO presented lower forward scatter (186.33 ± 9.33 and 196.89 ± 26.86, respectively) compared to NoCRYO-0h (322.80 ± 3.36), indicating that cryopreservation reduced nuclei size. Thus, a simple method for cryopreservation and isolation of adipocyte nuclei causing less damage to DNA integrity was proposed.