Patients

In the present study, 15 patients were included with deletions in the proximal long arm of chromosome 3. One patient, case 2, was previously described clinically and cytogenetically by Ogilvie et al 1998,5 while the remaining 14 patients are novel. Clinical information was systematically collected from clinicians, using supplementary table 1.

The WHO Child Growth Standards were used to standardise birth height, weight, and occipitofrontal circumference (OFC) for all novel patients and for the previously reported patients where growth parameters were given.19 WHO standards are available up to 19 years of age for height and up to 10 years of age for weight. To assess OFC after birth the German head circumference references were used, which extend up to 18 years of age.20

The clinical investigations and genetic analyses were performed according to the guidelines in the Declaration of Helsinki and were approved by the ethics committee of Uppsala University. Informed consent was obtained from all family members and specific permission to publish photographs was obtained.

Molecular details

We present the clinical and molecular features of 15 novel patients harbouring deletions of the proximal long arm of chromosome 3. One of the patients was reported cytogenetically in the late 1990s5 (case 2). The present study also provides a review of 13 previously reported patients.1–14 The deletions are mapped within 3q12.3q21.3 and they range in size from the smallest of 580 kb (case 13) to the largest of 22.4 Mb (case 4) (figure 1 and table 1). Most of the deletions have different breakpoints, although the breakpoints in cases 9, 10, 11, and 12 are in close proximity (figure 1); the breakpoints of these patients are approximately located at 113.5–116.5 Mb. In all but one case, the deletion was the sole identified aberration. In case 7, an inversion was identified (inv(3)(q13.1q26.3)) and the deletion was located at the 3q13.1 inversion breakpoint. The deletions showed a de novo occurrence in 13 cases. Parental DNA was not available for testing in case 8, whose 7.2 Mb deletion is likely to have arisen de novo because of the size and gene content of this deleted region. Carrier testing in the parents of case 14 was only possible in the mother's DNA, which revealed a normal chromosome 3.

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Figure 1

A physical map of the chromosomal region 3q11.2 to 3q23, illustrating the deletions. The deletions identified in novel patients are shown in black, previously reported deletions that have been molecularly characterised are shown in dark grey, and previously reported deletions that have been cytogenetically characterised are shown in light grey. RefSeq genes are indicated in blue. The grey solid box illustrates the shortest region of overlapping deleted region, and a zoomed view shows the five RefSeq genes within this region (bottom panel).

The shortest region of overlapping deletion (SRO) is delineated by case 13, with estimated breakpoints at genomic positions 115.33—115.39 Mb (figure 1). This 580 kb segment includes five RefSeq genes: DRD3, ZNF80, TIGIT, MIR568, and ZBTB20. The SRO is shared by 13 of the novel patients, and by 11 of the previously reported patients—that is, 24 cases in total. The SRO is within the previously reported smallest deletion which was 1.9 Mb in size.13 However, the 2.5 Mb deletion identified in the case presented by Simovich et al is located at 3q13.11q12 and hence does not overlap with the region defined in the present study.14 Four cases do not have an overlapping deletion with the SRO, namely case 14, case 15 (both from this study), Kosaki et al,10 and Simovich et al.14

Clinical data

In total, clinical data from 28 patients, both novel and previously published cases, were collected and are summarised in supplementary table 1. Photographs of some of the novel patients (cases 1, 2, 4, 5, 6, 7, 9, 12 and 15) are shown in figure 2. The clinical findings in the 24 patients sharing the SRO (115.33—115.39 Mb) are summarised separately, and the frequency of these features was calculated (supplementary table 1, frequency column). These features include normal pregnancy and delivery at term with a few exceptions.1 4 5 11 14 Developmental delay is the most prevalent feature, present in 19/21 cases. Two cases did not suffer from developmental delay (case 7) or had not been examined at the time of the report due to the patient's young age (case 4). However, case 7 displayed attention deficit disorder. There are eight patients presenting with autism or attention deficits and one with epilepsy, including case 7. In 15 of 17 cases speech was delayed, and in three of these 15 patients the speech was minimal/no meaningful words were used/communication by hands by the age of 4.5, 8, and 18 years. Muscular hypotonia was found in 12/15 patients. Interestingly, muscular hypotonia was suggested by Shimojima et al to be the only common finding along with developmental delay in patients with 3q13 deletions13. The brain and central nervous system were also affected: five patients had agenesis of the corpus callosum, three patients had ventriculomegaly, and one had alobar holoprosencephaly. In total, seven patients displayed skull malformations: two with dolichocephaly, two with plagiocephaly, and three with brachycephaly. Furthermore, 10/13 patients presented with broad and prominent forehead.

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Figure 2

Photographs of cases 1, 2, 4, 5, 6, 7, 9, 10, 12, and 15. Physical characteristics of note are short philtrum, protruding lips with full lower lips and tented upper lips, hypertelorism, and antimongoloid slanted eyes present in several cases.

Distinct recognisable facial features, including short philtrum, protruding lips with full lower lips and tented upper lips, antimongoloid slanted eyes, and hypertelorism, were apparent in several cases (figure 2). In total, the facial features in the 24 patients were short philtrum in 6/6, epicanthal folds in 8/14, hypertelorism in 7/17, antimongoloid slant in 7/13, a high arched palate in 7/10, and ptosis in 4/11. Ocular malformation included strabismus in 6/14 and myopia in 4/8. The ears were large in 5/15 and were low set in 4/15.

There was a high prevalence of abnormal external male genitalia, affecting 11/15 males. The abnormalities included micropenis (4/15), microorchidism (2/15), cryptorchidism (7/15), and shawl scrotum (2/15). All female patients had normal genitalia.

Skeletal malformations were a frequent finding, present in as many as 16/24 patients. The skeletal malformations included scoliosis, lordosis, thoracic kyphosis, joint contractures, and peripheral malformations affecting the hands and feet. Of note were the proximally set thumbs present in three novel cases (cases 2, 4, and 9).

Growth parameters were assessed in the 24 patients sharing the SRO and 4/11 (cases 4, 5, 9, and 10) had a birth OFC >85th centile (7/11 had a birth OFC >50th centile). OFC was also available at a later age and 9/20 had an OFC >85th centile (11/20 had an OFC >50th centile). Case 6 is noteworthy, having a birth OFC between 15–50th centile and an OFC between 85–97th centile at the age of 4 years and 10 months. The weight and length parameters were also reviewed and these were normal at birth, 5/16 had a weight >50th centile, 5/12 had a length >50th centile, and none of the patients displayed a weight or length >85th centile. At the time of report, 10/19 had a weight >50th centile and 9/19 had a weight >85th centile. Regarding height at the time of report, 13/21 were >50th centile and 10/21 were >85th centile. Hence, a postnatal growth pattern above the mean was observed among these patients. A larger region, encompassing 18.2 Mb in q13.11q13.33, has previously been identified in a screening of patients with syndromic overgrowth, and the present report delineates the overgrowth candidate region to 3q13.31. In DECIPHER, most of the listed microdeletion/microduplication syndromes are associated with short stature, while there is one that is characterised by tall stature—the 15q26 overgrowth syndrome. Regarding OFC, there is one listed microdeletion/microduplication syndrome in DECIPHER with macrocephaly, the 1q21.1 microduplication syndrome, in comparison with microcephaly that is present in 13 of the DECIPHER listed syndromes. Known overgrowth syndromes are Sotos syndrome, Beckwith—Wiedeman syndrome, Simpson—Golabi—Behmel syndrome, Klinefelter syndrome, homocystinuria and Marfan syndrome.21 The molecular knowledge about overgrowth syndromes is thus fairly limited and, in this context, the present report provides novel clues to finding genes involved in growth.

Candidate genes

The proximal long arm of chromosome 3 is a gene dense region (figure 1) with 145 genes within the estimated boundaries (chr3:103.32–128.18 Mb) of the 15 novel patients. Hence, a number of genes could potentially contribute to the phenotypic features of these patients. Regarding the five RefSeq genes present in the SRO, two (DRD3 and ZBTB20) are particularly interesting with respect to developmental delay, the neuropsychiatric features, and the structural brain, central nervous system, and skull malformations. DRD3 encodes D3 subtype of the dopamine receptors, which is localised to the limbic areas of the brain, and is involved in locomotion, cognition, emotion, and affection as well as neuroendocrine secretion.22 Targeted mutation of DRD3 is associated with hyperactivity in mice, and recent association studies in patients with neuropsychiatric disorders have explored the contribution of DRD3 variants to their phenotype.23–25 The ZBTB20 gene belongs to the BTB/POZ zinc finger family and is expressed in the developing hippocampal neurons.26 Downregulation of ZBTB20 disturbs the normal maturation of a certain type of neurons in the hippocampus, and changes in the cortical cytoarchitecture—for example, lack of the posterior part of the corpus callosum—were observed in transgenic mice models.27 28 One additional interesting aspect of ZBTB20, with respect to the observed postnatal overgrowth in the patients, is the fact that it regulates genes involved in growth and metabolism.29

In addition to the SRO and the genes therein, the present study provides clues about other 3q genomic regions harbouring important genes with respect to normal development. First, the deletion in case 14, telomeric of the SRO, contains LSAMP and GAP43, two strong candidate genes for developmental delay. LSAMP encodes the limbic system associated membrane protein, and studies in both human and mice models have demonstrated the involvement of LSAMP in neuropsychiatric features and behaviour.30 31 GAP43 is involved in neurite outgrowth, neurotransmission, and synaptic plasticity among other functions and GAP43 was also recently identified as a candidate gene for autism and autistic-like manifestations in human and mice.32–34 In addition, Gap43 +/− mice display decreased corpus callosum and hippocampal commissure volume.34 Secondly, the present study supports the previous suggestion that 3q11 could harbour a locus for agenesis of the corpus callosum (ACC).2 35 Five previously published 3q deletion patients exist who displayed ACC.2 4 5 7 35 Here we present one novel patient (case 6) with ACC, having a deletion that can help with further refining of the ACC critical region. As discussed above, strong candidate genes involved in ACC are ZBTB20 and GAP43.

Further support underlining the importance of DRD3, ZBTB20, LSAMP, and GAP43 in contributing to the phenotype in patients with 3q13 deletions is their haploinsufficiency score, as defined by the study by Huang et al.36 There are 49 genes of the total of 145 genes in 3q12-q21 that have a haploinsufficiency score of <50%, and these four candidate genes are among those 49 (supplementary table 2).