Mutation screening in Borjeson-Forssman-Lehmann syndrome: identification of a novel de novo PHF6 mutation in a female patient

Background

Börjeson‐Forssman‐Lehmann syndrome (BFLS; MIM 301900) is an infrequently described X linked disorder caused by mutations in PHF6, a novel zinc finger gene of unknown function.

Objective

To present the results of mutation screening in individuals referred for PHF6 testing and discuss the value of prior X‐inactivation testing in the mothers of these individuals.

Results

25 unrelated individuals were screened (24 male, one female). Five PHF6 mutations were detected, two of which (c.940A→G and c.27_28insA) were novel. One of these new mutations, c.27_28insA, was identified in a female BFLS patient. This was shown to be a de novo mutation arising on the paternal chromosome. This is the first report of a clinically diagnosed BFLS female with a confirmed PHF6 mutation. In addition, the X‐inactivation status of the mothers of 19 males with suggested clinical diagnosis of BFLS was determined. Skewed (⩾70%) X‐inactivation was present in five mothers, three of whom had sons in whom a PHF6 mutation was detected. The mutation positive female also showed skewing.

Conclusions

The results indicate that the success of PHF6 screening in males suspected of having BFLS is markedly increased if there is a positive family history and/or skewed X‐inactivation is found in the mother.     

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents

Background

The underlying causes of mental retardation remain unknown in about half the cases. Recent array‐CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal.

Objective and methods

Array‐CGH with approximately 3500 large insert clones spaced at ∼1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals.

Results

Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits.

Conclusions

In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH‐microarray for guiding genetic counselling.     

AUNX1, a novel locus responsible for X linked recessive auditory and peripheral neuropathy, maps to Xq23-27.3

Background

We report here the genetic characterisation of a large five generation Chinese family with the phenotypic features of auditory neuropathy and progressive peripheral sensory neuropathy, and the genetic feature of X linked recessive inheritance. Disease onset was at adolescence (at an average age of 13 years for six affected subjects). The degree of hearing impairment varied from mild to severe, with decreased otoacoustic emissions; auditory brainstem responses were lacking from onset.

Methods

Two‐point and multipoint model based linkage analysis using the MILNK and LINKMAP programs of the FASTLINK software package produced maximum two‐point and multipoint LOD scores of 2.41 and 2.41, respectively.

Results

These findings define a novel X linked auditory neuropathy locus/region (AUNX1, Xq23–q27.3). This region is 42.09 cM long and contains a 28.07 Mb region with flanking markers DXS1220 and DXS8084, according to the Rutgers Combined Linkage‐Physical Map, build 35. However, mutation screen of the candidate gene SLC6A14 within the region did not identify the causative genetic determinant for this large Chinese family.     

A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network

Background

Fanconi anaemia is a heterogeneous genetic disease, where 12 complementation groups have been already described. Identifying the complementation group in patients with Fanconi anaemia constitutes a direct procedure to confirm the diagnosis of the disease and is required for the recruitment of these patients in gene therapy trials.

Objective

To determine the subtype of Fanconi anaemia patients in Spain, a Mediterranean country with a relatively high population (23%) of Fanconi anaemia patients belonging to the gypsy race.

Methods

Most patients could be subtyped by retroviral complementation approaches in peripheral blood T cells, although some mosaic patients were subtyped in cultured skin fibroblasts. Other approaches, mainly based on western blot analysis and generation of nuclear RAD51 and FANCJ foci, were required for the subtyping of a minor number of patients.

Results and conclusions

From a total of 125 patients included in the Registry of Fanconi Anaemia, samples from 102 patients were available for subtyping analyses. In 89 cases the subtype could be determined and in 8 cases exclusions of common complementation groups were made. Compared with other international studies, a skewed distribution of complementation groups was observed in Spain, where 80% of the families belonged to the Fanconi anaemia group A (FA‐A) complementation group. The high proportion of gypsy patients, all of them FA‐A, and the absence of patients with FA‐C account for this characteristic distribution of complementation groups.Fanconi anaemia is a rare hereditary recessive disease characterised by developmental abnormalities, bone marrow failure and predisposition to cancer, mainly acute myeloid leukaemia.¹ To date, 12 complementation groups have been reported (FA‐A, B, C, D1, D2, E, F, G, I, J, L and M) and 11 associated genes have already been identified: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, BRIP1/FANCJ, FANCL and FANCM/Hef.^{2,3,4,5,6,7,8,9,10,11,12,13,14} In dividing cells or in cells exposed to DNA damage, eight Fanconi anaemia proteins (FANCA/B/C/E/F/G/L/M) form a Fanconi anaemia core complex, necessary for the monoubiquitination of FANCD2.^2,5,15 In contrast with these Fanconi anaemia proteins, FANCD1 and FANCJ are not involved in FANCD2 monoubiquitination, indicating that these proteins participate downstream of FANCD2 in the FA/BRCA pathway.^2,7Because of the overlap in the phenotype and molecular pathways between the different chromosome fragility syndromes, Fanconi anaemia subtyping constitutes an invaluable approach to confirm the diagnosis of the disease.^16,17 Additionally, in the case of patients with FAD1, subtyping analysis allows the identification of BRCA2 mutation carriers, characterised by an increased risk of developing breast, ovarian and other types of cancers.¹⁸ Fanconi anaemia subtyping also facilitates mutation screening studies and therefore the identification of mutations with particular pathogenic effects. In addition to the above‐mentioned applications, subtyping is essential before enrolling a patient with Fanconi anaemia in a gene therapy trial.Progress in the cloning of Fanconi anaemia genes enabled the identification of mutations in specific Fanconi anaemia genes by means of DNA sequencing approaches or other methods.¹⁹ The large number and complexity of some Fanconi anaemia genes and their mutations, together with the necessity of verifying the pathogenicity of each new mutation, implies that subtyping of patients with Fanconi anaemia by mutational analysis is often time consuming and laborious. The possibility of reverting the phenotype of Fanconi anaemia cells by the transfer of functional Fanconi anaemia genes has been recently proposed as an efficient approach for identifying the pathogenic genes that account for the disease in patients with Fanconi anaemia.^20,21 A different Fanconi anaemia subtyping approach is based on the western blot analysis of FANCD2.²² By means of the observation of the ubiquitinated (FANCD2‐L) and non‐ubiquitinated (FANCD2‐S) forms of the protein FANCD2, it is possible to predict pathogenic mutations in proteins upstream or downstream of FANCD2.²³ In the case of patients belonging to rare complementation groups such as FAD1 or FA‐J, approaches based on the formation of RAD51 or BRIP1 nuclear foci are also highly informative in identifying their complementation group.²⁴With the purpose of determining the prevalence of the different Fanconi anaemia complementation groups in Spain, we conducted an extensive subtyping study of Fanconi anaemia in this Mediterranean country. In addition to a predominantly caucasian population, a relatively large population of about 500 000 gypsies also live in Spain. In this population, the incidence of recessive syndromes is high, owing to the high rates of consanguinity.²⁵ This study will allow us to identify potential differences in the distribution of Fanconi anaemia subtypes due to geographical and ethnic characteristics, and will also allow us to conduct further mutation studies within the population of patients subtyped for Fanconi anaemia. Additionally, our subtyping study will facilitate the enrolling of patients with Fanconi anaemia in clinical gene therapy trials aimed at the genetic correction of their haematopoietic stem cells.     

Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Løken syndrome

Background

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that constitutes the most common genetic cause of renal failure in the first three decades of life. Using positional cloning, six genes (NPHP1‐6) have been identified as mutated in NPHP. In Joubert syndrome (JBTS), NPHP may be associated with cerebellar vermis aplasia/hypoplasia, retinal degeneration and mental retardation. In Senior–Løken syndrome (SLSN), NPHP is associated with retinal degeneration. Recently, mutations in NPHP6/CEP290 were identified as a new cause of JBTS.

Methods

Mutational analysis was performed on a worldwide cohort of 75 families with SLSN, 99 families with JBTS and 21 families with isolated nephronophthisis.

Results

Six novel and six known truncating mutations, one known missense mutation and one novel 3 bp pair in‐frame deletion were identified in a total of seven families with JBTS, two families with SLSN and one family with isolated NPHP.     

Sensorineural deafness and male infertility: a contiguous gene deletion syndrome

Background

Syndromic hearing loss that results from contiguous gene deletions is uncommon. Deafness‐infertility syndrome (DIS) is caused by large contiguous gene deletions at 15q15.3.

Methods

Three families with a novel syndrome characterised by deafness and infertility are described. These three families do not share a common ancestor and do not share identical deletions. Linkage was established by completing a genome‐wide scan and candidate genes in the linked region were screened by direct sequencing.

Results

The deleted region is about 100 kb long and involves four genes (KIAA0377, CKMT1B, STRC and CATSPER2), each of which has a telomeric duplicate. This genomic architecture underlies the mechanism by which these deletions occur. CATSPER2 and STRC are expressed in the sperm and inner ear, respectively, consistent with the phenotype in persons homozygous for this deletion. A deletion of this region has been reported in one other family segregating male infertility and sensorineural deafness, although congenital dyserythropoietic anaemia type I (CDAI) was also present, presumably due to a second deletion in another genomic region.

Conclusion

We have identified three families segregating an autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility. This new syndrome is caused by the deletion of contiguous genes at 15q15.3.     

Disruption of an exon splicing enhancer in exon 3 of MLH1 is the cause of HNPCC in a Quebec family

Background

A 3 bp deletion located at the 5′ end of exon 3 of MLH1, resulting in deletion of exon 3 from RNA, was recently identified.

Hypothesis

That this mutation disrupts an exon splicing enhancer (ESE) because it occurs in a purine‐rich sequence previously identified as an ESE in other genes, and ESEs are often found in exons with splice signals that deviate from the consensus signals, as does the 3′ splice signal in exon 3 of MLH1.

Design

The 3 bp deletion and several other mutations were created by polymerase chain reaction mutagenesis and tested using an in vitro splicing assay. Both mutant and wild type exon 3 sequences were cloned into an exon trapping vector and transiently expressed in Cos‐1 cells.

Results

Analysis of the RNA indicates that the 3 bp deletion c.213_215delAGA (gi:28559089, {"type":"entrez-nucleotide","attrs":{"text":"NM_000249.2","term_id":"28559089","term_text":"NM_000249.2"}}NM_000249.2), a silent mutation c.216T→C, a missense mutation c.214G→C, and a nonsense mutation c.214G→T all cause varying degrees of exon skipping, suggesting the presence of an ESE at the 5′ end of exon 3. These mutations are situated in a GAAGAT sequence 3 bp downstream from the start of exon 3.

Conclusions

The results of the splicing assay suggest that inclusion of exon 3 in the mRNA is ESE dependent. The exon 3 ESE is not recognised by all available motif scoring matrices, highlighting the importance of RNA analysis in the detection of ESE disrupting mutations.     

Size bias of fragile X premutation alleles in late-onset movement disorders

Background

Fragile X‐associated tremor/ataxia syndrome (FXTAS), caused by premutation expansions (55–200 CGG repeats) of the FMR1 gene, shares clinical features with other movement disorders, particularly in the domains of gait ataxia, intention tremor and parkinsonism. However, the prevalence of FXTAS within other diagnostic categories is not well defined.

Methods

A meta‐analysis was conducted of all published (n = 14) genetic screens for expanded FMR1 alleles to assess the prevalence and CGG‐repeat size bias of FMR1 premutation alleles in those populations.

Results

In men with late‐onset cerebellar ataxia, the prevalence of premutation alleles (1.5%; 16/1049) was 13 times greater than expected based on its prevalence in the general population (2%; 16/818 for age of onset >50 years; odds ratio 12.4; 95% confidence interval 1.6 to 93.5). Meta‐analysis of CGG‐repeat data for screened patients with premutation alleles shows a shift to larger repeat size than in the general population (p<0.001). 86% (19/22) of premutation alleles were larger than 70 repeats in the patients screened, whereas only approximately 22% of premutation alleles are larger than 70 repeats in the general population.

Conclusions

Expanded FMR1 alleles contribute to cases of late‐onset sporadic cerebellar ataxia, suggesting that FMR1 genetic testing should be carried out in such cases. The biased distribution of FMR1 allele sizes has substantial implications for genetic counselling of carriers with smaller alleles who are at a low risk of developing FXTAS, and suggests that the estimated prevalence of FXTAS among men >50 years of age in the general population may be two to threefold lower than the initial figure of 1 in 3000.     

Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems

Background

Heterozygous mutations in the COL1A1 or COL1A2 gene encoding the α1 and α2 chain of type I collagen generally cause either osteogenesis imperfecta or the arthrochalasis form of Ehlers‐Danlos syndrome (EDS). Homozygous or compound heterozygous COL1A2 mutations resulting in complete deficiency of the proα2(I) collagen chains are extremely rare and have been reported in only a few patients, albeit with variable phenotypic outcome.

Methods

The clinical features of the proband, a 6 year old boy, were recorded. Analysis of proα and α‐collagen chains was performed by SDS‐polyacrylamide gel electrophoresis using the Laemmli buffer system. Single stranded conformation polymorphism analysis of the proband''s DNA was also carried out.

Results

In this report we show that complete lack of proα2(I) collagen chains can present as a phenotype reminiscent of mild hypermobility EDS during childhood.

Conclusions

Biochemical analysis of collagens extracted from skin fibroblasts is a powerful tool to detect the subset of patients with complete absence of proα2(I) collagen chains, and in these patients, careful cardiac follow up with ultrasonography is highly recommended because of the risk for cardiac valvular problems in adulthood.     

CRYM mutations cause deafness through thyroid hormone binding properties in the fibrocytes of the cochlea

Background

In a search for mutations of μ‐crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C‐terminus in patients with non‐syndromic deafness.

Objective

To investigate the mechanism of hearing loss caused by CRYM mutations

Methods

T3 binding activity of mutant μ‐crystallin was compared with that of wild‐type μ‐crystallin, because μ‐crystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where μ‐crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody.

Results

One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that μ‐crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K‐ATPase.

Conclusions

CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. μ‐Crystallin may be involved in the potassium ion recycling system together with Na,K‐ATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K‐ATPase, T3, and deafness.     

Genotype phenotype correlation of 30 patients with Smith-Magenis syndrome (SMS) using comparative genome hybridisation array: cleft palate in SMS is associated with larger deletions

Background

Smith‐Magenis syndrome (SMS) is rare (prevalence 1 in 25 000) and is associated with psychomotor delay, a particular behavioural pattern and congenital anomalies. SMS is often due to a chromosomal deletion of <4 Mb at the 17p11.2 locus, leading to haploinsufficiency of numerous genes. Mutations of one of these gemes, RAI1, seems to be responsible for the main features found with heterozygous 17p11.2 deletions.

Methods

We studied DNA from 30 patients with SMS using a 300 bp amplimers comparative genome hybridisation array encompassing 75 loci from a 22 Mb section from the short arm of chromosome 17.

Results

Three patients had large deletions (10%). Genotype–phenotype correlation showed that two of them had cleft palate, which was not found in any of the other patients with SMS (p<0.007, Fisher''s exact test). The smallest extra‐deleted region associated with cleft palate in SMS is 1.4 Mb, contains <16 genes and is located at 17p11.2‐17p12. Gene expression array data showed that the ubiquitin B precursor (UBB) is significantly expressed in the first branchial arch in the fourth and fifth weeks of human development.

Conclusion

These data support UBB as a good candidate gene for isolated cleft palate.     

The C20orf133 gene is disrupted in a patient with Kabuki syndrome

Background

Kabuki syndrome (KS) is a rare, clinically recognisable, congenital mental retardation syndrome. The aetiology of KS remains unknown.

Methods

Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution.

Results

In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain, but also in kidney, eye, inner ear, ganglia of the peripheral nervous system and lung.

Conclusion

The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous.     

Congenital hyperinsulinism and mosaic abnormalities of the ploidy

Background

Congenital hyperinsulinism and Beckwith‐Wiedemann syndrome both lead to β islet hyperplasia and neonatal hypoglycaemia. They may be related to complex genetic/epigenetic abnormalities of the imprinted 11p15 region. The possibility of common pathophysiological determinants has not been thoroughly investigated.

Objective

To report abnormalities of the ploidy in two unrelated patients with congenital hyperinsulinism.

Methods

Two patients with severe congenital hyperinsulinism, one overlapping with Beckwith‐Wiedemann syndrome, had pancreatic histology, ex vivo potassium channel electrophysiological studies, and mutation detection of the encoding genes. The parental genetic contribution was explored using genome‐wide polymorphism, fluorescent in situ hybridisation (FISH), and blood group typing studies.

Results

Histological findings diverged from those described in focal congenital hyperinsulinism or Beckwith‐Wiedemann syndrome. No potassium channel dysfunction and no mutation of its encoding genes (SUR1, KIR6.2) were detected. In patient 1 with congenital hyperinsulinism and Beckwith‐Wiedemann syndrome, paternal isodisomy for the whole haploid set was homogeneous in the pancreatic lesion, and mosaic in the leucocytes and skin fibroblasts (hemihypertrophic segment). Blood group typing confirmed the presence of two erythroid populations (bi‐parental v paternal only contribution). Patient 2 had two pancreatic lesions, both revealing triploidy with paternal heterodisomy. Karyotype and FISH analyses done on the fibroblasts and leucocytes of both patients were unremarkable (diploidy).

Conclusions

Diploid (biparental/paternal‐only) mosaicism and diploid/triploid mosaicism were present in two distinct patients with congenital hyperinsulinism. These chromosomal abnormalities led to paternal disomy for the whole haploid set in pancreatic lesions (with isodisomy or heterodisomy), thereby extending the range and complexity of the mechanisms underlying congenital hyperinsulinism, associated or not with Beckwith‐Wiedemann syndrome.     

Genotype-phenotype correlations of 39 patients with Cornelia De Lange syndrome: the Dutch experience

Background

Cornelia de Lange syndrome (CdLS) is a multiple congenital anomaly syndrome characterised by a distinctive facial appearance, prenatal and postnatal growth deficiency, psychomotor delay, behavioural problems, and malformations of the upper extremities. Recently mutations in NIPBL, the human homologue of the Drosophila Nipped‐B gene, were found to cause CdLS. Mutations have been found in 39% of reported cases.

Methods

Patients were enrolled in the study and classified into one of four groups based on clinical examination: classic, mild, possible, or definitively not CdLS. Three dimensional photography was taken of 20 subjects, and compared between groups. Behaviour was assessed with specific attention to autism. We searched for mutations in NIPBL and correlated genotype with phenotype.

Results

: We found mutations in 56% of cases.

Conclusions

Truncating mutations were generally found to cause a more severe phenotype but this correlation was not absolute. Three dimensional facial imaging demonstrated the potential for classifying facial features. Behavioural problems were highly correlated with the level of adaptive functioning, and also included autism. No correlation of behaviour with the type of mutation was found     

Stability of the m.8993T->G mtDNA mutation load during human embryofetal development has implications for the feasibility of prenatal diagnosis in NARP syndrome

Background

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Because of the high transmission risk and the absence of therapy in these disorders, at‐risk couples often ask for prenatal diagnosis (PND). However, because heteroplasmy load (coexistence of mutant and wild‐type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases.

Methods

We performed 13 prenatal diagnoses for the NARP (neurogenic weakness, ataxia, retinitis pigmentosa) m.8993T→G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocyte samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts.

Results

Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were consistently mutation‐free. In 8/13 PND, mutant load was <30%. These children are healthy at 2–7 years of age. In 5/13 PND, mutant load ranged from 65 to 100%, and parents preferred to terminate the pregnancies (15–22 weeks of gestation). Single‐cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and amniocyte mutant load, despite striking intercellular variation. The m.8993T→G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 terminations, respectively, were identical in all tissues from a given individual (mean (SD) 78 (1.2)%, 91 (0.7)%, 74 (2)%, and 63 (1.6)% for the 4 fetuses, respectively).

Conclusions

Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. Although these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help in interpreting PND results.     

Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation

Introduction

Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder that is usually associated with mutations in the MECP2 gene. The most common mutations in the gene are p.R168X and p.T158M. The influence of X‐chromosome inactivation (XCI) on clinical severity in patients with RTT with these mutations was investigated, taking into account the extent and direction of skewing.

Methods

Female patients and their parents were recruited from the UK and Australia. Clinical severity was measured by the Pineda Severity and Kerr profile scores. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes, and allele‐specific polymerase chain reaction was used to determine the parental origin of mutation. Combining these, the percentage of cells expected to express the mutant allele was calculated.

Results

Linear regression analysis was undertaken for fully informative cases with p.R168X (n = 23) and p.T158M (n = 20) mutations. A statistically significant increase in clinical severity with increase in the proportion of active mutated allele was shown for both the p.R168X and p.T158M mutations.

Conclusions

XCI may vary in neurological and haematological tissues. However, these data are the first to show a relationship between the degree and direction of XCI in leucocytes and clinical severity in RTT, although the clinical utility of this in giving a prognosis for individual patients is unclear.Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder, usually caused by mutations in the methyl‐CpG‐binding protein 2 (MECP2, OMIM#300005) gene. Mutations often arise at CpG hotspots,¹ and the most common mutations in MECP2 found in RTT cases are p.T158M and p.R168X (RettBASE, http://mecp2.chw.edu.au/). RTT has a wide clinical variability in terms of its severity.² Studies investigating the association between genotype and phenotype were originally quite inconsistent in their findings. However, with larger studies and increasing numbers of publications, evidence for definite relationships between genotype and phenotype is becoming clearer.³ Apparent differences in study results often occurred because of the use of different means of classifying and recording clinical severity. Additionally, the effects of X‐chromosome inactivation (XCI) status and other epigenetic influences on MECP2 function are likely to have a real influence on the variation in phenotype associated with specific mutations.XCI occurs early in embryogenesis at the blastula stage, and is usually a random process.^4,5 The inactive X chromosome is determined as cells become pluripotent; once this has happened, lineages derived from each of these cells will all have the same X chromosome inactivated through a process of methylation. Some studies have shown that there is an increased tendency for skewing of XCI in lymphocytes in RTT when compared with age‐matched controls, and that this usually confers a protective effect.^6,7,8 The most striking clinical examples of the effects of XCI in RTT are seen in twins with disparate severity^9,10 and in healthy carrier mothers with skewed XCI (presumed favourable) with affected daughters.^{11,12,13,14,15}In this study, we adopted a new approach to investigate genotype–phenotype relationships in RTT by exploring the association between clinical severity and the proportion of active mutated allele for the two common MECP2 mutations, p.R168X and p.T158M.     

CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene

Background

CHARGE syndrome is a non‐random clustering of congenital anomalies including coloboma, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear anomalies, and deafness. A consistent feature in CHARGE syndrome is semicircular canal hypoplasia resulting in vestibular areflexia. Other commonly associated congenital anomalies are facial nerve palsy, cleft lip/palate, and tracheo‐oesophageal fistula. Specific behavioural problems, including autistic‐like behaviour, have been described. The CHD7 gene on chromosome 8q12.1 was recently discovered as a major gene involved in the aetiology of this syndrome.

Methods

The coding regions of CHD7 were screened for mutations in 107 index patients with clinical features suggestive of CHARGE syndrome. Clinical data of the mutation positive patients were sampled to study the phenotypic spectrum of mutations in the CHD7 gene.

Results

Mutations were identified in 69 patients. Here we describe the clinical features of 47 of these patients, including two sib pairs. Most mutations were unique and were scattered throughout the gene. All patients but one fulfilled the current diagnostic criteria for CHARGE syndrome. No genotype‐phenotype correlations were apparent in this cohort, which is best demonstrated by the differences in clinical presentation in sib pairs with identical mutations. Somatic mosaicism was detected in the unaffected mother of a sib pair, supporting the existence of germline mosaicism.

Conclusions

CHD7 mutations account for the majority of the cases with CHARGE syndrome, with a broad clinical variability and without an obvious genotype‐phenotype correlation. In one case evidence for germline mosaicism was provided.