Comparison of X-chromosome inactivation patterns in multiple tissues from human females.

BACKGROUND: X-chromosome inactivation (XCI) is the mechanism by which gene dosage uniformity is achieved between female mammals with two X chromosomes and male mammals with a single X chromosome, and is thought to occur randomly. For molecular genetic testing, accessible tissues (eg blood) are commonly studied, but the relationship with inaccessible tissues (eg brain) is poorly understood. For accessible tissues to be informative for genetic analysis, a high degree of concordance of genetic findings among tissue types is required. OBJECTIVE: To determine the relationship among multiple tissues within females at different ages (fetus to 82 years). METHODS: XCI patterns were analysed using the polymorphic androgen receptor (AR) gene assay. DNA was isolated from 26 different human females without history of malignancy, using 34 autopsy tissues representing the three embryonic germ layers. RESULTS: 33 of the 280 tissue samples analysed from 13 of the 26 females showed skewed XCI values (>80:20%). Average XCI value was not significantly different among the tissues, but a trend for increasing XCI variability was observed with age in blood and other tissues studied (eg the SD for all tissues studied for the 0-2 years group was 9.9% compared with 14.8% in the >60 years group). We found a significant correlation (r(s) = 0.51, p = 0.035) between XCI values for blood and/or spleen and brain tissue, and in most other tissues representing the three embryonic germ layers. CONCLUSIONS: In our study, XCI data were comparable among accessible (eg blood) and inaccessible tissues (eg brain) in females at various ages, and may be useful for genetic testing. A trend was seen for greater XCI variability with increasing age, particularly in older women (>60 years).

Novel splice isoforms for NLGN3 and NLGN4 with possible implications in autism.

OBJECTIVE: To screen cDNA for NLGN3 and NLGN4 from lymphoblastoid cells from autistic subjects. METHODS AND RESULTS: 10 young autistic females and 30 non-autistic subjects were studied for alterations in two X linked genes, NLGN3 and NLGN4. A novel NLGN4 isoform lacking exon 4, which occurred de novo on the paternal allele, was identified in one of the autistic females. Monoallelic expression of NLGN4 was seen in this subject and in 11 of 14 informative autistic and non-autistic females using a single nucleotide polymorphism found at 3' UTR. Additionally, the NLGN3 transcript was present in two isoforms (with and without exon 7) in nine of 10 autistic females and in 30 non-autistic subjects, including parents of the autistic female having only the complete transcript with exon 7, and from the whole brain of a control. The novel truncated NLGN3 product may have a regulatory role, as reported in other proteins (for example, vasopressin receptor) by attenuating the function of the full length isoform, resulting in a reduction of the mature protein. Three dimensional protein structures were characterised using comparative modelling, and significant changes were suggested in the protein cores for these two neuroligin isoforms. CONCLUSIONS: Splice variants may lead to potentially abnormal neuroligins in the causation of autism spectrum disorders.

Brief report: non-random X chromosome inactivation in females with autism.

Autism is a heterogeneous neurodevelopmental disorder with a 3-4 times higher sex ratio in males than females. X chromosome genes may contribute to this higher sex ratio through unusual skewing of X chromosome inactivation. We studied X chromosome skewness in 30 females with classical autism and 35 similarly aged unaffected female siblings as controls using the polymorphic androgen receptor (AR) gene. Significantly, increased X chromosome skewness (e.g., >80:20%) was detected in our autism group (33%) compared to unaffected females (11%). X chromosome skewness was also seen in 50% of the mothers with autistic daughters. No mutation was seen in the promoter region of the XIST gene reported to be involved in X chromosome inactivation in our subjects. X chromosome skewness has been reported in female carriers of other neurological disorders such as X-linked mental retardation, adrenoleukodystrophy and Rett syndrome.

Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations.

The genetic aetiology of autism remains elusive. Occasionally, individuals with Cowden syndrome (a cancer syndrome) and other related hamartoma disorders such as Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and Proteus-like conditions, are characterised by germline PTEN mutations, and may have neurobehavioural features resembling autism as well as overgrowth and macrocephaly. Therefore, we undertook PTEN gene mutation analysis in 18 subjects mainly prospectively ascertained with autism spectrum disorder and macrocephaly. Of these 18 autistic subjects (13 males and five females; ages 3.1-18.4 years) with a head circumference range from 2.5 to 8.0 standard deviations above the mean, three males (17%) carried germline PTEN mutations. These three probands had previously undescribed PTEN mutations: H93R (exon 4), D252G (exon 7), and F241S (exon 7). They had the larger head circumference measurements amongst all our study subjects. The three residues altered in our patients were highly evolutionarily conserved. We suggest that PTEN gene testing be considered for patients with autistic behaviour and extreme macrocephaly. The gene findings may impact on recurrence risks as well as medical management for the patient.

Insulin resistance and obesity-related factors in Prader-Willi syndrome: comparison with obese subjects.

Prader-Willi syndrome (PWS), the most common genetic cause of marked obesity in humans, is usually due to a de novo paternally derived chromosome 15q11-q13 deletion or maternal disomy 15 [(uniparental disomy (UPD)]. Obesity is due to energy imbalance, but few studies have examined fat patterning and obesity-related factors in subjects with PWS (deletions and UPD) compared with subjects with simple obesity. We examined for differences in fatness patterning and lipid, leptin, and glucose and insulin levels in subjects with simple obesity and PWS and adjusted for gender, age, and body mass index (BMI). Fasting peripheral blood samples and cross-sectional magnetic resonance image scans at the level of the umbilicus were obtained in 55 subjects ranging in age from 10.4 to 49 years: 20 PWS deletion, 17 PWS UPD, and 18 obese controls. Subcutaneous fat area (SFA) and intra-abdominal visceral fat area (VFA) were calculated. No significant difference was seen between the PWS deletion subjects or PWS UPD subjects for fatness measurements or leptin levels. Twenty-three of 37 PWS subjects met the criteria for obesity (BMI > 95th percentile). No significant differences were observed for SFA and VFA between the PWS subjects judged to be obese and control subjects with simple obesity. There was an overall trend for decreased VFA in the PWS subjects but not significantly different. VFA was significantly positively correlated with both fasting insulin and total cholesterol in PWS deletion subjects but not in PWS UPD subjects or obese controls. Fasting insulin level was significantly lower in the obese PWS subjects compared with subjects with simple obesity, and insulin sensitivity (QUICKI) was significantly higher in PWS subjects with obesity. Homeostasis model assessment (HOMA) and QUICKI values were correlated and in opposite directions with triglycerides in the obese PWS subjects but not in the obese controls. Subjects in each group were stratified according to published criteria on the basis of their level of visceral fat (e.g. > or = 130 cm(2)) to assess the influence of VFA on metabolic abnormalities. In the obese PWS subjects, the fasting triglyceride, glucose, and insulin levels, and HOMA value were significantly elevated, while the QUICKI value was significantly lower in those with VFA > or = 130 cm(2). Such significant differences were not seen in the obese control group. Our results indicate that VFA may be regulated differently in PWS subjects compared to individuals with simple obesity. Insulin resistance is lower in PWS subjects and insulin sensitivity is higher compared with obese controls. PWS subjects with increased VFA may be at a higher risk of obesity-related complications compared to PWS subjects without increased VFA.

Microarray analysis of gene/transcript expression in Prader-Willi syndrome: deletion versus UPD.

BACKGROUND: Prader-Willi syndrome (PWS), the most common genetic cause of marked obesity, is caused by genomic imprinting and loss of expression of paternal genes in the 15q11-q13 region. There is a paucity of data examining simultaneous gene expression in this syndrome. METHODS: We generated cDNA microarrays representing 73 non-redundant genes/transcripts from the 15q11-q13 region, the majority within the PWS critical region and others distally on chromosome 15. We used our custom microarrays to compare gene expression from actively growing lymphoblastoid cell lines established from nine young adult males (six with PWS (three with deletion and three with UPD) and three controls). RESULTS: There was no evidence of expression of genes previously identified as paternally expressed in the PWS cell lines with either deletion or UPD. We detected no difference in expression of genes with known biallelic expression located outside the 15q11-q13 region in all cell lines studied. There was no difference in expression levels of biallelically expressed genes (for example, OCA2) from within 15q11-q13 when comparing UPD cell lines with controls. However, two genes previously identified as maternally expressed (UBE3A and ATP10C) showed a significant increase in expression in UPD cell lines compared with control and PWS deletion subjects. Several genes/transcripts (for example, GABRA5, GABRB3) had increased expression in UPD cell lines compared with deletion, but less than controls indicating paternal bias. CONCLUSIONS: Our results suggest that differences in expression of candidate genes may contribute to phenotypic differences between PWS subjects with deletion or UPD and warrant further investigations.

Novel mutation in the KCNQ4 gene in a large kindred with dominant progressive hearing loss.

Analysis of genotyping of a five-generation American family with nonsyndromic dominant progressive hearing loss indicated linkage to the DFNA2 locus on chromosome 1p34. This kindred consists of 170 individuals, of which 51 are affected. Pure tone audiograms, medical records, and blood samples were obtained from 36 family members. Linkage analysis with five microsatellite markers spanning the region around DFNA2 produced a lod score of 6.6 for the marker MYCL1 at straight theta = 0.0. Hearing loss in this family showed a very similar pattern as the first reported American family with the same linkage. High frequency hearing loss was detectable as early as 3 years of age, and progressed to severe to profound loss by the fourth decade. Using intronic primers, we screened the coding region of the KCNQ4 gene. Heteroduplex analysis followed by direct sequencing identified a T-->C transition at position 842, which would produce an L281S amino acid substitution. The observed mutation was shown to segregate completely with affected status in this family. The L281 residue is significantly conserved among the other members of the voltage-gated K(+) channel genes superfamily. Hydrophobicity analysis indicated that L281S substitution would lower formation of the beta structure at the P region of this ion channel. Mutation analysis of KCNQ4 was also performed on 80 unrelated probands from families with recessive or dominant nonsyndromic hearing loss. None of these cases showed a truncated mutation in KCNQ4.