Delineation variable genotype/phenotype correlations of 6q27 terminal deletion derived from dic(6;18)(q27;p10)

Background Terminal deletion of 6q27 produces a rare syndrome associated with unexplained mental retardation, hypotonia, epilepsy, and multiple malformations. Structural brain malformations are consistently observed, including agenesis of the corpus callosum, hydrocephalus, periventricular nodular heterotopia, polymicrogyria, and cerebellar malformations. Here we report a fetal risk assessment of a 27-year-old woman with mental retardation, hypotonia and dysmorphic features at 17 weeks of pregnancy. Results Cytogenetic analyses revealed an addition at chromosome 6qter in the mother. Haploinsufficiency of 6q27 to 6qter (1.3 Mb) and trisomy of the entire short arm of chromosome 18 (15.2 Mb) were found using a single nucleotide polymorphism based array. Results were confirmed by molecular cytogenetics and multiplex ligation-dependent probe amplification. The karyotype of the mother was 46,XX dic(6;18)(6pter → 6q27::18p10 → 18pter).arr [hg19]6q27(169,591,548-170,898,549) × 1,18p11.3p10(12,842-15,375,878) × 3.ish dic(6;18)(q27;p10)(RP11-614P3-,RP11-1035E2+,D18Z1+). Deletion of 6q27 was associated with the structural brain malformations, whereas trisomy of 18p had minor clinical effects. The unbalanced rearrangement of chromosome 6 and chromosome 18 was de novo and was not inherited by the developing fetus. Conclusions A rare rearrangement between 6q27 and 18p was identified, which led to a de novo 1.3 Mb deletion of 6q27 and a 15.2 Mb duplication of 18p in an adult with mental retardation, hypotonia, epilepsy, and multiple malformations.

Here, we report a smallest 6q27 deletion with trisomy18p in a 27-year old patient at 17 weeks gestation. The patient presented with severe mental retardation (MR), hypotonia, and other malformations ( Table 1). The goals of this report was to elucidate the genotype/ phenotype correlation and test the hypothesis that the deletion results from unbalanced chromosome translocation and inheritance.

Case presentation
The patient was the product of the first pregnancy of non-consanguineous parents. Both parents and younger brother were healthy, and the family history lacked evidence of mental retardation (MR) and hypotonia. The patient was delivered at 41 weeks gestation. During the entire pregnancy, the mother of the patient felt sick and experienced emesis. Antiabortifacients were taken from 18 weeks of gestation until birth. The patient presented intra-uterine growth retardation; birth weight was approximately 1,500 g (>3SD), and birth length was 40 cm (>2SD); she also exhibited a weak cry and suckling. No apparent congenital abnormalities were noticed, although global developmental delays were observed.
At age two, the patient had few single words and walked with an unstable gait. At age 27, evaluation revealed an IQ score of 46, hypotonia, prominent forehead, hypertelorism, long philtrum and, slow nystagmus, ataxic gait, joint laxity, bilateral clinodactyly, talipes cavus, scoliosis, pelvic obliquity, genu valgum on right, and femur length discrepancy of 1 cm ( Figure 1A).

Cytogenetic and fluorescence in situ hybridization (FISH) analyses
Chromosome karyotype analyses showed 46,XX,add(6) (q27) ( Figure 3A). Her parental and fetal karyotypes were normal. FISH confirmed the deletion of 6q27 and duplication of 18p including parts of the centromere ( Figure 3C).

Multiplex ligation-dependent probe amplification (MLPA) and real-time qPCR analyses
MLPA P036-E2 was used to confirm the deletion of 6q27 and duplication of 18p ( Figure 4A). Meanwhile, haploinsufficiency of the C6orf70 gene, related to the clinical phenotype of 6q27 deletion syndrome, was confirmed by qPCR ( Figure 4B). From these data, the karyotype of the patient was revised to 46,XX,dic (  RP11-1036E2+,D18Z1+).The risk of recurrence for the further pregnancy was 50%.

Discussion
Here we report a patient with MR in the 17th week of pregnancy. Standard cytogenetic analyses revealed additional chromosomal material at the end of chromosome 6. SNP-Array analyses detected a 1.3 Mb deletion of 6q27-qter and a 15.2 Mb duplication of the entire short arm of chromosome 18. Result from the SNP-Array were confirmed by FISH and MLPA. Deletion of 6q27, ranging from 0.4 Mb to 10.8 Mb, can result from unbalanced translocation or isolated deletion and produce a rare syndrome [1][2][3][4][5]. Partial terminal monosomy of 6q27 produces brain structural abnormalities, dysmorphic features, developmental delay, epilepsy, learning difficulties, and hypotonia. Patients with distal 6q27 deletions have been described, revealing variable brain phenotypes including agenesis of the corpus callosum (ACC), periventricular nodular heterotopias (PNH), cerebellar malformations, polymicrogyria, and hydrocephalus. Previous studies have shown that terminal 1.7 Mb of 6q27 harbors four genes, THBS2, PHF10, DLL1, and C6orf70 that play critical roles in morphogenesis of the nervous system during embryogenesis [4].
ACC is associated with many syndromes and shows significant variability ranging from complete absence or partial absence to hypoplasia of the corpus callosum. Twelve genomic loci are consistently associated with ACC, including the 6q terminal deletion [8]. Incidence of corpus callosum anomalies for 6q25-27 deletion is approximately 50% to 75%. The subject of this study presented with mild hypogenesis, a short, comma-shaped corpus callosum ( Figure 1B). PNH lining the lateral ventricles due to defective neuronal migration and can by asymptomatic or present as small, unilateral or bilateral nodules, or as extensive agglomerates, with or without other brain abnormalities.
C6orf70 in the 6q27 region is a critical gene expressed during brain development in human and rodents. The gene product is a putative vesicle-associated protein that plays a major role in controlling neuronal migration. Haploinsufficiency or mutation of C6orf70 was reported to cause PNH [1], and incidence of PNH was 9 out of 12 patients of 6q25-27 deletion. Although PCR analyses confirmed the patient in this report had haploinsufficiency of C6orf70, no evidence of PNH was found, suggesting that haploinsufficiency of C6orf70 for PNH has variable phenotypic expression.
Other brain malformations, such as cerebellar malformation, polymicrogyria, and hydrocephalus, have also been reported in 6q27 deletion cases with variable penetrance and expression. Cerebellar malformations were reported in 10.8% and polymicrogyria in 6.7% of individuals. Interestingly, neither of these conditions were present in the patient characterized in this study, although colpocephaly, asymmetric lateral ventricles, and choroid fissure cysts on the left were present. These results may suggest that the same genes cause various different brain malformations through the same pathway. It is also possible that low resolution MRI may have missed subtle brain malformations.
Mental retardation and seizures are associated with brain malformation in 6q27 deletion patients. The TATA-binding Figure 2 Size, extent, and genomic content of deletions including 6q27 in cases comparable to the present one. All patients had structural brain abnormalities. Using this information for mapping of a critical region of brain malformations revealed C6orf70, PHF10, DLL1, and TBP as putative candidate genes for structural brain malformations.
protein (TBP) gene in 6q27 seemed the most likely causal mutation [3]. TATA-binding protein, a general transcription factor associates with aggregates in several polyglutamine disorders [9]. Although trisomy18p alone is tolerated in humans and presents as very mild mental retardation [6][7], it is reasonable to speculate that severe MR (IQ = 46) in the subject may result from the combination of TBP deletion and trisomy18p.
Hypotonia is a clinical phenotype that affects approximately half of 6q27 deletion patients. Brain malformation may cause this phenotype, and long-lasting repercussions of hypotonia without corrective procedures, include scoliosis, pelvic obliquity, genu valgum, and leg discrepancy. In this case study, chromosome 6 is dicentric, one centromere is from chromosome 6 and the other is from chromosome 18. When human chromosome are dicentrics or multicentrics, faulty alignments may result [10]. Three different 'predominant' activation patterns occur. When the distance between two centomeres is close, they are fused and operate together. When the distance between centromeres is 1.4 Mb to 13 Mb, both centromeres are active, and when the distance is over 15 Mb, only one is active [11]. In this case, the distance between the centromeres was estimated to be more than 15 Mb, indicating only one centromere was active.
Assessment of risk for the offspring was a major goal of this study. Results indicated no genetic abnormalities were present in the amniotic fluid, and the fetus was developmentally normal. The baby was born with no complications.

Conclusions
In conclusion, this study identified a rare rearrangement between 6q27 and 18p, which led to de novo 1.3 Mb

HumanCytoSNP-12 BeadChip for CNVs and SNP analyses
DNA was analyzed with the Illumina Human CytoSNP-12 array to 100 kb resolution following the instructions provided in the Illumina Infinium® HD Assay Ultra manual. The iScan scanner was used to translate electronic signals into digital signals. Initial analyses and quality control were performed using Illumina GenomeStudio software. Copy number variation was determined using the Illumina KaryoStudio software.

MLPA and real-time qPCR analyses
P036-E2 probe mix contains a probe designed to detect deletions and duplications for every subtelomeric region. MLPA reactions were performed following the manufacturer's instructions (Biometra Thermal Cycler, Westburg, Netherlands). Fragment separation was performed using the ABI-3130 sequencer (Applied Biosystems, Foster City, CA), and data was directly analyzed using Coffalyser software. Primers for the C6orf70 gene were designed for use in real-time qPCR analyses on an ABI 7900 HT fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA). Primers sequences and PCR conditions are available by request.