Validation of Single Nucleotide Variants Potentially Associated with Schizophrenia Susceptibility in 22q11.

2 Deletion Patients
Samantha E.
1, Mammen

Elisabeth E.

1, Mlynarski

Beverly S.

1 Emanuel

1. The Childrens Hospital of Philadelphia, Philadelphia, PA

Abstract syndrome that occurs in Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic
approximately 1 out of 4000 live births. The deletion can arise de novo or can be inherited as an autosomal dominant disorder. Common clinical findings of patients with 22q11DS include cardiac anomalies, immune system deficiencies, palatal defects and increased risk for neuropsychiatric disorders. Specifically, 22q11DS patients have about a 30% chance of developing schizophrenia while the morbid risk for the general population is slightly less than 1%. We hypothesize that the higher susceptibility for schizophrenia is due to a single nucleotide variant s, mutations or a combination of variants located on the non-deleted allele of chromosome 22. For this project, the non-deleted allele of chromosome 22 was deep sequenced in fifty five patients with 22q11DS, three of whom were diagnosed schizophrenics. A list of twelve possibly damaging, homozygous variants was compiled by PolyPhen, one of which was located in SNAP29, a gene that is mutated in CEDNIK syndrome. I validated the presence of these variants in the relevant patients and tested additional patients for the presence of SNAP29 mutations. This work confirmed the presence of variants identified through deep sequencing, and indicates that there is no damaging SNAP29 mutation common to 22q11DS.

Function of SNAP29
SNAP29 is a gene that codes for a SNARE protein involved in vesicle fusion It is important for vesicle exo- and endocytosis Proteins encoded from SNAP29 operate by slowing down the recycling of fusion machinery and down regulating synaptic vesicle turnover Patients with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome) have been found to have a 1 base pair deletion in SNAP29. It is an autosomal recessive disorder. SNAP29 is a gene in the deleted region of 22q11. 22q11DS patients are missing one copy of the gene and a mutation in the other copy might be expected to give rise to CEDNIK syndrome.

Sequencing of child with cleft lip and SNAP29 variant and her parent

Sequencing of other patients with the cleft lip phenotype

Methods
PCR was used to amplify the DNA of individual patients The success of the PCR was checked via agarose gel electrophoresis Amplified DNA was sequenced using Sanger sequencing at CHOPs NAPCore facility 4Peaks and Geneious computer software programs were used to visualize and edit individual DNA sequences

Validation of Identified Variants

Location of Possibly and Probably Damaging Single Nucleotide Variants

We first sequenced the mother (D03-086) of the child with the SNAP29 variant to determine if the SNP was inherited The variant is not present in the mothers DNA No DNA was available for the father

The only significant phenotype of the patient with the SNAP29 change was a cleft lip We sequenced two other patients with cleft lips and did not find the SNAP29 21224655 variant

Sequencing of SNAP29 Exon 2 ARVCF 19978218

ARVCF 19965512
The SNAP29 21224655 variant is found in Exon 2 of the gene We sequenced Exon 2 in 35 22q11DS patients, including the two with the cleft lip phenotype Three examples of this sequencing are shown above We did not find any SNAP29 variants in any of the patients

DGCR8 rs118025402

CLTCL1 19198017

Sequencing of SNAP29 Exon 1

CLTCL1 rs712952

CLTCL1 rs807459

MED15 20937201

DGCR14 19130247
The one base pair deletion seen in CEDNIK is found in Exon 1 of SNAP29 We are in the process of sequencing Exon 1 of the 35 patients who had Exon 2 sequenced So far, we have completed sequencing of 7 patients The only variant we have found so far is SNAP29 rs1061064, which is a silent mutation found in about 40% of the population

CLTCL1 rs1061325

GNB1L 19130247

Conclusions
12 out of 12 (100%) of the indentified variants were validated HIC2 21800227 was found to be heterozygous We have not found any connection between cleft lip and SNAP29 so far. Out of the 7 patients who have had Exon 1 of SNAP29 sequenced, we have not found any potentially damaging abnormalities In the future, we will finish sequencing Exon 1 of SNAP29 in the remaining 28 patients and look for the SNAP29 21224655 variant in more patients with the cleft lip phenotype

References
HIC2 21800227
1. Sprecher E, Ishida-Yamamoto A, Mizrahi-Koren M, Rapaport D, Goldsher D, Indelman M, Topaz O, Chefetz I, Keren H, OBrien T, Bercovich D, Shalev S, Geiger D, Bergman R, Horowitz M, Mandel H. A Mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma. Am. J. Hum. Genet. 2005, 77:242-251 2. McDonald-McGinn D, Sullivan K. Chromosome 22q11.2 Deletion Syndrome (DiGeorge Syndrama/Velocardiofacial Syndrome). Medicine. 2011, 90:1-18 3. McDermid H, Morrow B. Genomic Disorders on 22q11. Am. J. Hum. Genet. 2002, 70:1077-88 4. Arinami T. Analyses of the associations between the genes of 22q11 deletion syndrome and schizophrenia. J. Hum. Genet. 2006, 51:1037-1045

Acknowledgments
Emanuel Lab
Colleen Franconi Sarah Tash Takema Kato Meghan McNamara

CHOP
NAPCore CRISSP faculty CRISSP staff Elaine Zackai Donna McDonaldMcGinn Genetic clinical staff

SNAP29 21224655

Emory
Stephen Warren Joshua Suhl

Penn
Raquel Gur BBL Team