Introduction

Chromosome 6p deletions are rare events within the population. At present, there are 43 cases in the medical literature, excluding ring chromosome 6 anomalies. These include 27 reports on terminal deletions of 6p, six cases on unbalanced translocations, and the remaining 10 cases describe interstitial deletions with the entire 6p22, 6p22.2–p25.2 or 6p24–p25 segment deleted1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 (see references therein).

The clinical findings vary in the interstitial deletion 6p syndromes, and include orofacial clefting, short neck, clinodactyly or syndactyly and brain, heart and kidney defects. In contrast, corneal opacity, iridogoniodysgenesis anomaly, various Rieger type anomalies, hypertelorism and deafness are associated with 6p terminal deletions.

A large number of mouse model studies have provided valuable insights into the function of genes located in the syntenic segment of mouse chromosome 13, including roles in organogenesis, and in particular eye, cardiac and skeletal development.12, 13, 14, 15, 16, 17

In this paper, we describe six new cases that enable us to delineate the 6p deletion syndrome involving the 10 Mbp long 6p24–pter chromosomal segment with further accuracy. Our findings allow us to associate developmental defects (see listing in Discussion) to deletions of specific chromosomal segments and to candidate genes therein.

Patient 1

The youngest of two siblings (DOB 08/11/95) was born after an uneventful pregnancy and delivery: the patient was male, with a birth weight of 3.6 kg (50th centile), head circumference was 49.8 cm (25th–50th centiles) and height was on the 25th centile.

Symptoms at birth

Hypertelorism and down-slanting palpebral fissures.

Initial cytogenetic data

G-banded preparations showed that the subtelomeric segment was absent from the derivative chromosome 6. Subsequent in situ hybridisation confirmed the initial karyotype: 46,XY,del(6)(p25.1). This event is de novo as both parents' karyotypes were normal.

Additional clinical data

Ophthalmolgical assessment revealed strabismus (which recurred after surgery) and marked posterior embryotoxon of both eyes. Anterior fibres from the iris adhered to the embryotoxon in places, but the anterior chamber was otherwise normally formed. The intra ocular pressure was normal.

The patient's ears were slightly low set and posteriorly rotated (Figure 1). A CT scan of the ears showed a poorly pneumatised mastoid on the left side. He had a broad nasal bridge, a highly arched palate, abnormal dentition.

Figure 1
figure 1

Photographs of five of the 6p deletion cases; (a) Patient 1; (b) Patient 2; (c) Patient 3; (d) Patient 5; (e) Patient 6.

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Physical examination at 5 years noted a harsh systolic and a very soft early diastolic murmur after the aortic component of the second sound. Echocardiography demonstrated a minor structural abnormality of his aortic valve, though it was functioning normally.

He also had a left-sided cervical skin tag, slightly broad toes and his audiometry revealed bilateral mild to moderate low-frequency sensorineural hearing loss. He had moderate learning difficulties, language delay and motor milestones were delayed, as he sat at 11 months and walked at around approximately 16 months. A Griffith's assessment (14-01-99) showed him to be a year behind his chronological age of 4 years (see Table 1 for summary of phenotypes).

Table 1 Main characteristics of chromosome 6p deletion cases presented in this study
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Patient 2

Female patient (DOB 3/05/00) was born to a 29-year-old woman after a full-term delivery, complicated by abruptio placenta requiring emergency caesarean. Birth weight was 3.2 kg (45th centile).

Symptoms at birth

Hypotonia, facial dysmorphism, abnormal creases in the hand and feet and hyperflexion of both feet.

Initial cytogenetic data

Cytogenetic analysis using G-banded chromosomes revealed the karyotype: 46,XX, del(6)(p24). This event is de novo as both parents' karyotypes were normal.

Additional clinical data

At 20 months, she was treated for pneumonia and had been diagnosed with asthma.

Examination at 2 years revealed a prominence in the occipital area, hypertelorism, hypermetropia and a strabismus (Figure 1). The teeth were abnormal and low in enamel. She had a carped shaped mouth, a small patent ductus arteriosus (PDA) and patent foramen ovale (PFO). Both feet had a rocker bottom appearance with bilateral ankle pronation. Hypotonia and joint hyperlaxity conditions were still present.

Gross motor skills were judged to be at 15 months level as she was not able to walk unassisted at 2 years. Adaptive skills were judged to be at 16–19 months level, cognitive skills were normal at 2 years. Skeletal development was showing delay and the anterior fontanel had not closed. She displayed hypersensitivity to temperature, auditory hypersensitivity and the mouth showed poor response to stimulation. A CT scan revealed a prominence in the fourth ventricle with questionable changes in the cerebella vermis and cerebella white matter.

In contrast to the parents, who are Hispanic, the baby was very fair. Thyroid stimulating hormone (TSH) levels were high, a blood test indicated high immunoglobulin E (IgE) levels and diagnosed anaemia. She was undergoing occupational and speech therapy in addition to physiotherapy.

Her proprioceptor input showed hyperactive irregularity and she displayed seizures that occur with a frequency of around 60 per day lasting for up to 1 min per seizure. It is thought these seizures were non specific and can occur in children with a developmental delay (see Table 1 for summary of phenotypes).

Patient 3

A girl, the eldest of two siblings (DOB 13-01-84), was born after an uneventful pregnancy and delivery, with a birth weight of 4.0 kg (>90th centile). The parents are non consanguineous and show no apparent family history of malformations. She presented at the age of 1 year with developmental delay and concerns about her hearing and vision.

Initial cytogenetic data

At 5 years, her chromosomes were analysed and a de novo del (6) p25 → pter karyotype was identified.

Additional clinical data

Examination at 13 years revealed macrocephaly (head circumference 56.5 cm), upslanting short palpebral fissures, prominent epicanthic folds, mild dysmorphic features and a short philtrum. Eye examination revealed telecanthus, anterior segment dysgenesis, a divergent squint, hypermetropia, and mild pigmentary retinopathy (Figure 1). The teeth were prominent and overcrowded. Her toes were clawed, with a short second toe and bilateral fifth finger clinodactyly. She also had hip dysplasia, diagnosed as bilateral Perthes disease. She had undergone tonsillectomy, adenoidectomy, insertion of grommets and had previously suffered from faecal soiling secondary to severe constipation and overflow diarrhoea. In addition, she suffered from conductive hearing loss, with impaired motor skills and slow speech.

Patient 4

A second child, female was born to healthy nonconsanguineous parents (DOB 14-12-00), with a birth weight of 3. 7 kg (60th centile).

Symptoms at birth

Examination at birth showed her to have hydrocephalus. She had been diagnosed with Dandy–Walker Syndrome with possible agenesis of the corpus callosum. A ventriculoperitoneal shunt had been inserted at 6 weeks of age.

Initial cytogenetic data

G-banded chromosome analysis revealed her to be: 46,XX, del (6)(p24.3 → pter): in situ hybridisation also confirmed this karyotype. The parents' metaphase chromosomes were also analysed, both were normal for the respective region, indicating a de novo event.

Additional clinical data

Corneal opacities consistent with Peter's anomaly: she received bilateral cornea transplants at 2 weeks of age. A ventricular septal defect (VSD) was diagnosed and subsequently repaired at age 4 weeks. A gastrointestinal tube was inserted for feeding at approximately 6 weeks of age (see Table 1 for summary of phenotypes).

Mild sensorineural hearing loss in the right ear (35 db) and moderate to severe hearing loss in the left ear (60 db). Assessment at around 2 months indicated neurodevelopmental delay.

Patient 5

A female (DOB 02/04/02) was born to a 33- and 36-year-old mother and father, respectively. This was their first child and there was no clinical history of genetic abnormalities. Patient 5 was born at full term via a Caesarean section due to a breech presentation, with a birth weight of 3.54 kg (50th centile).

Symptoms at birth

Features present at birth included hypertelorism, a short neck, and a severely displaced hip: the latter condition possibly attributable to the breech presentation. Cerebral magnetic resonance imaging (MRI) revealed no significant abnormalities. Eye examinations, hearing tests and sonograms revealed no other abnormalities. The palate was highly arched. MRI detected some communication in the anterior flax. This was felt to have no clinical significance.

Initial cytogenetic data

Cytogenetic examination using G-banded chromosomes revealed a 46,XX,del(6)(p24.3) karyotype.

Additional clinical data

By 1 month of age, she had extensive hemangiomas and telangiectasias on the right arm and right shoulder girdle. A check up at 13 weeks showed occipital flattening, and mildly low set ears, mild pectus excavatum and mild hypotonia were present; radiographic examination showed the hips had healed. Patient 5 had suffered from numerous episodes of bronchiolitis and ear infections. A check up at 5 months revealed blue sclerae, hypotonia and loose joints (Figure 1). There was moderate motor delay, but otherwise, her growth, social and visual skills were good.

Examination of Patient 5 at 6 months showed relative brachycephaly and plagiocephaly appearance with right occipital bones somewhat more prominent than left, the anterior fontanel had not closed. The ears are posteriorly rotated and the nose was broad based. The abdomen was mildly protuberant with small fingertip-sized defect in the abdominal wall and umbilical hernia. The fingers were long and with ulnar deviation, the thumbs were bilaterally low set. The shoulder was particularly hyperflexible. The deep tendon reflexes were normal and symmetric throughout (see Table 1 for summary of phenotypes).

Patient 6

A first-born male (DOB 13-3-00) to a nonconsanguineous Hispanic mother and father of 23 and 28 years, respectively. The boy was born at 35 weeks gestation following treatment for preterm labour from 29 weeks.

Symptoms at birth

At birth, he had asymmetric flattening of the occiput, bilateral cleft lip, PDA, atrial septal defect (ASD); mild pelviectasis was noted on renal ultrasound and he had an imperforate anus. A brain stem auditory evoked response was normal. The bilateral cleft lip and imperforate anus were repaired in the first year.

Initial cytogenetic data

Cytogenetic analysis using G-banded chromosomes revealed a de novo interstitial deletion that was initially reported as: 46XY,del (6)(p23p24.3) de novo.ish 6pter (TelVysion probe 6p x 2).

Additional clinical data

At 7½ months, physical examination showed hypertelorism with prominent eyes, flat supraorbital ridges and grey-blue sclerae (Figure 1), small posteriorly rotated, mildly dysplastic ears with bilateral preauricular pits. The left ear was overfolded and the right ear lacked the superior ramus of the antihelix. He had frontal upsweep of hair, a broad nose with a bulbous tip, but the palate was intact. There was mild pectus excavatum, a small umbilical hernia and an imperforate anus. The penis was small and testes were in the scrotum. Mild eczema was present. The middle toes were overlapping and thumbs were often adducted. The digits were otherwise normal except for spoon-shaped toenails. Neurological examination was normal except for mild drooling. Growth and development were essentially normal. At 13 months, he was at the 90th percentile for height and the 25th percentile for weight. Head circumference was also normal at the 10–25th percentile. At 13 months, the patient was taking one or two steps independently and he could say at least four words. His developmental assessment was age appropriate (see Table 1 for summary of phenotypes).

Family history included heart defects present in maternal second-degree relatives.

Methods

Tissue culture and fluorescent in situ hybridisation (FISH) were performed as described previously.1 In total, 13 YAC, 44 PAC, two BAC clones and one cosmid, that had been previously FISH mapped to defined locations on 6p or contained identifiable genetic markers and genes, were used. The PAC clones were chosen from the Sanger Institute's FPC database (http://www.sanger.ac.uk/cgi-bin/humace/fpcwebmap, accessed May 2003); clones were ordered according to fingerprinting and marker content results. Clones present in Ensembl are a selection of the fingerprinted clones, and it is therefore possible to estimate the position of the clones used in this study in the Ensembl database (http://www.ensembl.org/, accessed May 2003). The YAC clones were ordered according to reported marker content by cross-referencing in both databases and confirmed by our own FISH-based clone ordering experiments. Additional data were compiled from the Ensembl database (May 2003) to describe genes identified so far on distal 6p with respect to their markers, known function, distance from the telomere and chromosomal localisation (see Table 2). Breakpoint positions in the cases described above are shown in Table 2 and reflect our best estimates.

Table 2 Data compiled from the Ensembl database (http://www.ensembl.org/), and the Sanger Institute's FPC database (see Methods for description)
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Results

Deletions were confirmed by FISH as terminal or interstitial using probes mapping to 6p23–pter (Table 3). A dense collection of probes was used around regions where breakpoints occurred (Table 3). Clones were denoted as positive when a hybridisation signal was detected on the derivative chromosome 6 [del (6)] and negative when no signal was present.

Table 3 List of probes used for in situ hybridisation on chromosomes of the 6p deletion cases
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Patient 1 has the smallest deleted 6p segment. YAC 952h4, which localises in 6p25.3, was positive on the respective del (6). However, PAC dJ223B1, which lies distal to the YAC, did not produce a hybridisation signal on del (6). The breakpoint lies between these probes: based on this result, we have refined the cytogenetic diagnosis and positioned the breakpoint in 6p25.2 (Table 3).

Patient 2 has the deletion breakpoint positioned in PAC DJ287K15, just distal of YAC 878B10, which produces a positive signal on del (6) and lies proximal in 6p25 (Table 3).

Patient 3 has the breakpoint positioned within the proximal segment of 6p25 with all probes distal to PAC dJ23O21 proving negative on del (6) (Table 3).

Patient 4 has the largest deleted segment with the breakpoint occurring within 6p24.3. PAC dJ436h5 did not hybridise on del (6), the probe lies just distal to BAC 648N19, which gave a positive signal on del (6) (Table 3). We were able to confirm the original cytogenetic diagnosis as 46XX,del (6)(p24.3).

PAC dJ118B18, which contains the FOXC1 gene and lies in the subtelomeric region of 6p, was used for FISH on chromosomes of patient 5. The results showed this distal region was indeed not deleted on del (6). The most proximal probe that did not hybridise on del (6) was PAC dJ133h11, this probe lies in 6p24.3, immediately distal to dJ167k8, that gave a positive signal on del (6) (Table 3). Thus, we were able to confirm the cytogenetic finding as 46XX,del (6)(p24.3p25.3).

The distal segment of 6p25 in Patient 6 was not deleted: instead, we have shown that the deletion is interstitial with BAC B18 marking the distal breakpoint and PAC 524G21 the proximal breakpoint (Table 3). Thus, we were able to refine the initial karyotype finding to 46XY,del (6)(p24p25.2). The extent of the deletion is less than previously thought from conventional cytogenetic methods.

Examples of FISH results of all cases are represented in Figure 2. Based on the FISH results and mapping data available from the Ensemble and the Sanger Centre's physical mapping database, the total amount deleted from chromosome 6 is: over 3.2 Mb from Patient 1, 5.8 Mb from Patient 2, 6 Mb from Patient 3, 9 Mb from Patient 4, 6.9 Mb from Patient 5 and 7 Mb from Patient 6. This corresponds to 0.09, 0.18, 0.17, 0.23, 0.17 and 0.14 % of the total human genome deleted, respectively. The phenotypes of the collective cases and corresponding photographs are shown in Table 1 and Figure 1, respectively.

Figure 2
figure 2

Examples of results showing metaphase chromosomes prepared from a selection of cases under investigation. Probes used for in situ hybridisation were labelled with biotin or digoxygenin and detected using fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate systems. Hybridisation signals are indicated by arrowheads. (Left) Mapping the terminal deletion of Patient 4; PAC 359L13 (mapping in 6p24, red) hybridised to both chromosome 6 and del (6), whereas the more distal Pac 90O12 (also mapping to 6p24, green) hybridised only to the normal chromosome 6. Therefore the breakpoint lies between these two clones within band 6p24. (Right) Mapping the interstitial deletion in Patient 5; PAC118B18(red), mapping in 6p25, hybridised to both chromosome 6 and del (6), and PAC 172H1 (green) hybridised proximal to PAC118B18 within 6p24 on normal chromosome 6p. A green signal derived from PAC 172H1 is also present on del (6), and overlaps with the red signal (PAC118B18) in 6p25, indicating that a significant proportion of the region distal to 6p24 and proximal to 6p25 has been deleted.

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Discussion

A number of genes have now been localised to the short arm of human chromosome 6, and current available data for this region have been analysed in an effort to gain direct relevance to our cases (Table 2). In addition, progress has been made in creating knockout mice for specific genes12, 17 or mice carrying defined chromosomal deletions.14 Phenotypic characterisation of these mice and comparison to human patients with relevant chromosomal deletions enables us to extract information about gene function in this region. The main phenotypes observed in the cases described here are discussed below:

Ocular defects

Our four 6p terminal deletion cases exhibit ocular abnormalities, sensorineuronal hearing loss and developmental delay (clinical findings that constitute the description of 6p terminal deletion syndrome).1 Although the extent of the deletion is different in each case, all are haploinsufficient for the region on 6p25 containing FOXC1 and associated with disorders of the eye, namely iridogoniodysgenesis, Axenfeld–Rieger anomaly/syndrome, primary congenital glaucoma and iris hypoplasia.18, 19 The variable penetrance of the eye anomalies observed in our 6p terminal deletion cases may be explained by different genetic backgrounds in agreement with the mouse model,14 but parental origin effects, stochastic developmental events or mutations in other genes of related function may contribute. In addition, the ocular finding of grey-blue sclerae (Patients 5 and 6) can be associated with deletion of the 6p24–p25 segment distal to AP-2 and proximal to FOXC1 (Tables 1 and 2).

Hydrocephalus

The mouse Mf1 (FOXC1) knockout model and the allelic ch mouse show pleiotropic skeletal and ocular phenotypes.12, 14 These mice on various genetic backgrounds present craniofacial defects, lethal congenital hydrocephalus, eye defects and abnormalities of the central nervous, urogenital and skeletal systems. Parts of these phenotypes, like the ocular defects, can occur in mice heterozygous for the Foxc1 mutation.15 Patient 4 presents with congenital hydrocephalus, heart and anterior eye developmental defects, showing considerable similarity to the congenital hydrocephalus (ch) mouse model. It would appear that by comparing our four terminal deletion cases (Table 3), hemizygosity for both Bone morphogenetic protein 6 (BMP6) and the FOXC1 gene produces such a phenotype as hypothesised by Kume et al.12 However, it is worth noting that the case reported by Navarro et al3 (and GK Mirza, AM Perle and J Ragoussis, unpublished results) and an unbalanced translocation case (CA in Davies et al1), present with hydrocephalus associated with the deletion of FOXC1, while BMP 6 is retained. Therefore, we can conclude that the factors determining the occurrence of hydrocephalus could be more complex than the contiguous deletion of the FOXC1-BMP6 chromosomal segment.

Heart defects

Patient 1 and one of our published 6p terminal deletion cases,1 carry deletions of FOXC1 and present with aortic valve abnormalities. Furthermore, aortic valve abnormalities have been diagnosed in the FOXC1 mouse model.13 Thus we conclude that FOXC1 hemizygosity appears to be an important determinant of aortic valve development. Suggestions that evolve from analysis of cases in the literature with molecular characterisations are that regions deleted proximal of 6p25 attribute to other heart defects, in particular ASD, VSD and PDA. In combination with Patients 5 and 6, we suggest the involvement of other loci positioned between 6p24.3 and 6p25 in heart development. Of the genes identified within the relevant chromosomal segment, BMP6 is a possible candidate since it is expressed in the heart.17

Bone formation

(a) Joint formation

Three cases presented with joint hyperlaxity; Patients 2, 5 and 3. The latter case presented with osteochondritis juvenilis deformans (Perthes disease). Additional reports, including the case described by Guillen-Navarro3 and an unbalanced translocation case (BD in Davies et al1), all with deletion of 6p25, present with delayed bone maturation and hip dislocations. In the case of Patients 2 and 3, these findings agree with the Mf1 knockout model; however, Patient 5 presents evidence for other genes further centromeric that contribute to chondrogenesis.

(b) Pectus excavatum

A further interesting aspect of Patients 5 and 6 is that the segment containing the BMP6 gene is deleted. Both cases exhibit pectus excavatum, consistent with the phenotypic finding in one of our previously reported cases carrying a deletion of BMP6.9 Mice lacking BMP6 (the heterozygotes are reported normal) show defects in the development of the sternum17 indicating that the human protein may have a similar function to the mouse homologue.

Hearing defects

Our 6p terminal deletion cases 1,3 and 4 present with hearing loss, and case 2 demonstrates auditory hypersensitivity. The interstitial deletion cases lack such symptoms and therefore we suggest that a gene involved in hearing development can be positioned distal to the most telomeric interstitial breakpoint observed (case 5), that is, distal to the region containing the GMDS gene (Tables 2 and 3).

Craniofacial anomalies

Our interstitial deletion cases, 5 and 6, are reported present with a highly arched palate and a cleft lip, respectively: the proximal breakpoints of both deletions lie in 6p24.3. A number of studies have shown linkage to this region for orofacial clefting.20, 21, 22 Patient 2 is the first case to have both cleft lip and palate and this could result from an interaction between loci in 6p24 and 6p25. In summary these cases support the evidence that genes contributing to orofacial clefting lie distal to AP2 and proximal to FOXC1 gene.

Skin defects

Haploinsufficiency for the desmoplakin (DSP) gene has been implicated in causing an inherited skin disorder, palmoplanter keratoderma. Patients 4, 5 and 6 are hemizygous for this gene. Only the latter case showed defects to the skin, namely eczema. Other related cases have reported excessively dry skin but none so far have reported palmoplantar keratoderma. We conclude that hemizygosity for DSP on its own is not sufficient to cause this disease.

Cognitive/neuromotor delay

With the exception of Patient 1, our cases are hemizygous for the gene neuritin (Table 2) and except for Patient 6, our cases present with either neuronal or motor development delay. Patient 2 presents with epilepsy. Neuritin is induced by neuronal activity and promotes neuritogenesis;23 therefore, haploinsufficiency for this gene may contribute to developmental delay in our cases.

Tooth development defects

Our terminal deletion patients, 1, 2 and 3, all show tooth abnormalities; Patients 5 and 6 have normal dentition. We can conclude that hemizygosity for the region between the most telomeric breakpoint in the vicinity of marker D6S1617 and the telomeric breakpoint of patient 6 results in such defects.

An important point that requires highlighting is that all cases have deletions that arose de novo. Patient 2 and our previous cases BD, GD, HH and SG have deletion breakpoints within the F13A gene.1 This part of the chromosome contains the fra (6) 6p25.1 common, aphidicolin-type fragile site, and it is of interest to fine map and characterise this site and to determine whether it is involved in the deletions presented here. The extent of deletion in our cases does not correlate with the severity of the phenotype (Tables 2 and 3b). Patient 1 has the most severe phenotype, but the least amount of deleted genome compared to the other five cases. Genetic background differences, parent-of-origin effects or undetected mosaicism may be responsible for the lack of correlation between phenotype severity and size of the deletions in our cases.

It is clear that the generation and detailed study of mouse models and human deletion cases have contributed significantly to understanding the molecular basis of chromosome 6p deletion syndromes. Further work involving generation of mutations in genes located within the segment syntenic to human chromosome 6p in mice will increase our understanding of the function and nature of developmental defects associated with these deletions (http://www.mgc.har.mrc.ac.uk/physical/del36h.html). Further details and links on 6p deletion cases are available at our website (http://www.well.ox.ac.uk/genomics).