J. Stephen Bamforth

Chromosome 1q21.1 Contiguous Gene Deletion Is Associated With Congenital Heart Disease

Congenital heart disease (CHD), comprising structural or functional abnormalities present at birth, is the most common birth defect in humans. Reduced expression of connexin40 (Cx40) has been found in association with atrial fibrillation, and deletion of Cx40 in a mouse model causes various structural heart abnormalities in 18% of heterozygotes. We screened 505 unrelated CHD cases for deletions or duplications of the Cx40 gene (GJA5) by real-time quantitative PCR, in order to determine whether altered copy number of this gene may be associated with a cardiac phenotype in humans. Dosage of Cx40 flanking genes (ACPL1 and Cx50 gene, GJA8) was determined by real-time PCR for all apparent positive cases. In total, 3 cases were found to carry deletions on chromosome 1q21.1 spanning ACPL1, Cx40, and Cx50 genes. Absence of heterozygosity was observed in all 3 index cases over a 1.5- to 3-Mb region. Samples from the parents of two cases were obtained, and microsatellites across 1q21.1 were genotyped. One of the apparently unaffected parents was found to carry this deletion. All 3 index cases presented with obstruction of the aortic arch as the common structural cardiac malformation, and had no consistent dysmorphic features. Genotyping of 520 unrelated normal controls for this deletion was negative. We hypothesize that this 1q21.1 multigene deletion is associated with a range of cardiac defects, with anomalies of the aortic arch being a particular feature.

Assessment of soft tissue facial asymmetry in medically normal and syndrome‐affected individuals by analysis of landmarks and measurements

We investigated soft tissue facial asymmetry in normal and syndrome-affected individuals ranging in age from 1 year to adulthood. The purposes of our study were to determine if facial asymmetry was greater in syndrome-affected individuals than in normal individuals and, if true, to distinguish those measurements that could be used in routine screening to identify the presence of syndromes in uncertain patients and, lastly, to investigate the causes of measurement asymmetry at the level of the landmarks. The last purpose was possible because we used a stereophotogrammetric method with which the three-dimensional (3D) landmark positions were obtained. In the statistically significantly different measurements, those from the right side were dominant, with one exception in each group, except normal males. In all groups the landmark analyses demonstrated the same trends, and while there was far less patterning in the 3D coordinates, these results were also consistent between the four groups. We compared the statistical findings of the 3D coordinates and measurements and found that there was no predictable relationship between significant findings in the landmarks and the measurements. In particular, we noted that statistical differences in measurements did not infer significant differences in the positions of the landmarks between the right and left sides of the face. Both the normal and syndrome-affected groups appeared to be equally canalized and similarly affected by developmental noise: When the bilateral measurement differences of each syndrome-affected subject were compared to the limits of normal asymmetry, less than 10% of the comparisons exceeded the norms.

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell‐of‐origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone‐rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner‐Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell‐of‐origin of retinoblastoma tumors and cells expressing CRX and OTX2.

Technical note: Different techniques, different results—a comparison of photogrammetric and caliper‐derived measurements

The primary goal of our study was to compare photogrammetric measurements with caliper-derived measurements. We also looked at the difference between caliper-derived measurements that were taken with and without the landmarks marked. Thirteen facial measurements were repeated ten times on two adult subjects as follows: 1) Calipers were used to take the measurements before the landmarks were marked on each subjects face; 2) the landmarks were then marked with a black pencil, and the calipers were used to take the measurements again; and 3) images were taken of each subject with the markings left on the face, and the measurements were extracted from these images. Compared with the caliper-derived data taken with the landmarks marked, the photogrammetric means and standard deviations were typically larger, leading us to conclude that there was a systematic difference between the data. The generally greater variation in the photogrammetric measurements was ascribed to poor conditions, such as shadows, oblique markings, and unmarked landmarks. When the data gathered by caliper with and without the landmarks marked were compared, a systematic difference was suggested by the number of statistically significant t-test probabilities. Marking the landmarks reduced the standard deviations in some measurements by controlling two sources of variation: differing pressure on the skin and slippage of the calipers. Anthropologists, medical geneticists, and others who use measurements for diagnostic or classificatory purposes should be aware that data gathered by different techniques may yield different results.

Genetic study of SOX9 in a case of campomelic dysplasia

Campomelic dysplasia (CD) is a sporadic autosomal dominant syndrome that results in skeletal malformation and developmental abnormalities. Death usually occurs neonatally as a result of respiratory insufficiencies, but life expectancy varies depending on the severity of the phenotype. XY sex reversal is common in CD, and a range of genital defects is observed in males and females. CD is due to mutations in SOX9, a member of the SOX (SRY-related HMG box) gene family. SOX9 is a transcription factor involved in chondrogenesis and sex determination. We present a CD patient with a normal 46,XX karyotype and female phenotype. Single-stranded conformation polymorphism analysis of DNA from this CD patient demonstrated a single-stranded conformation polymorphism shift in the C-terminal region of SOX9. DNA sequencing showed a frameshift mutation resulting from the insertion of a single guanine residue in nucleotide region 1,453-1,456. This insertion mutation creates a mutant SOX9 open reading frame that is 201 nucleotides longer than the normal gene. It has been shown that the C-terminal region of SOX9 is responsible for the transactivating ability of the protein. The frameshift identified here affects approximately half of the protein region needed for full transactivating function. We hypothesize that residual SOX9 function may explain why this patient survived infancy.

A New Case of Oculoectodermal Syndrome

An 11-month-old infant girl presented with right-sided features of aplasia cutis congenita of the scalp, unilateral epibulbar dermoids, eccentric pupil, coloboma of the right upper eyelid, and depigmentation of the fundus surrounding the right optic nerve. These findings were similar to the oculoectodermal syndrome reported by other clinicians and researchers.

RE: An epidemiological analysis of CHARGE syndrome: preliminary results from a Canadian study [Issekutz et al., 2005].

We were very interested to read the study of Issukutz et al. [2005]which showed an extremely high frequency of CHARGE syndrome of one in 8,500 live births in the three Atlantic provinces (New Brunswick, Nova Scotia, and Prince Edward Island) of Canada but no cases in our province (Alberta). We offer a few comments on the methodology of the Issekutz et al. study and also the absence of cases in Alberta. It is clear that the diagnosis of CHARGE cases in their study is almost certainly an accurate diagnosis given the expertise of the authors [Blake et al., 1998].While the authors do acknowledge the limitations of their study, we believe they have overlooked the most important point concerning the frequency of rare syndromes, namely, that to get an accurate estimate of the prevalence-at-birth one needs to have a much longer casefinding period than they had, i.e., 4 years, because random fluctuations can give a very distorted picture. For example, my colleagues and Ipublished twopapers on the relative frequency of Hurler and Hunter Syndromes (MPS1-H and MPS2) using the extensive and long term ascertainment studies of the BC Health Status Registry as well as the metabolic laboratory where all suspected cases would have been studied in the province ofBC [Lowry et al., 1990]. In theperiod 1952–1971we had eight cases of MPS1-H and six cases of MPS2. A follow-up study in 1990 disclosed only one new case of MPS1-H since 1971 and no MPS2 cases at all. We have outlined the reasons why we do not think that lack of ascertainment is the reason. Thus for Issekutz et al. [2005] we recommend a much longer case-finding period and considerable caution in interpreting their prevalence frequency. Another concern is the fact that their cases are derived from pediatricians who are members of the Canadian Paediatric Society (CPS), however, not all pediatricians may be members of that Society and furthermore inAlberta some casesmaywell have been referred by physicians other than pediatricians as none of the medical geneticists in the province is a member of the CPS. Issekutz et al’s paper does not disclose whether their cases were actually born in the province in which they were ascertained. We have examined the records of the Alberta Congenital Anomalies Surveillance System (ACASS) and have found two time periods with a number of cases and then a long gap with no cases. Between 1985 and 1989 inclusive we had seven cases giving a frequency of 1/30,610 followed by a gap of 2 years and then six cases in 1992 and 1993. Then from 1994 to 2000 there were no cases followed by one in each of the years 2001, 2003, and 2004. We do not yet have a denominator of births for 2004 but taking the period 1985 to 2003 we have 15 cases from 763,739 total births giving an overall frequency of 1/ 50,916 or approximately one in 51,000 in the 18 year period (13 ACASS cases supplemented by two further cases from the Edmonton Genetics Clinic). We have at least three other CHARGE cases whom we have not counted because they were born out of province although they are now residents in Alberta. We realize that there may be serious underascertainment of CHARGE cases in ACASS and are presently reviewing our cases of choanal atresia and coloboma which involve other anomalies. It is of interest to note that we have no gap in ascertainment of those two anomalies during the period when there were apparently no cases of CHARGE (1993–2000 inclusive). It is also quite possible that Dr. Blake’s interest and expertise has stimulated a greater awareness of the syndrome in her region which in turn translates into better ascertainment than in other regions. We look forward to further publications from her group.