Three-dimensional morphometric analysis of craniofacial shape in the unaffected relatives of individuals with nonsyndromic orofacial clefts: a possible marker for genetic susceptibility

Numerous studies have described altered patterns of craniofacial form in the unaffected relatives of individuals with nonsyndromic clefts. Unfortunately, results from such studies have been highly variable and have failed to provide a reliable method for differentiating "at-risk" relatives from controls. In the present study, we compared craniofacial shape between a sample of unaffected relatives (33 females; 14 males) from cleft multiplex families and an equal number of age/sex/ethnicity-matched controls. Sixteen x,y,z facial landmark coordinates derived from 3D photogrammetry were analyzed via Euclidean Distance Matrix Analysis, while 14 additional linear distances were analyzed via t tests. A subset of variables was then entered into a discriminant function analysis (DFA). Compared to controls, female unaffected relatives demonstrated increased upper facial width, midface reduction and lateral displacement of the alar cartilage. DFA correctly classified 70% of female unaffected relatives and 73% of female controls. Male unaffected relatives demonstrated increased upper facial and cranial base width, increased lower facial height and decreased upper facial height compared with controls. DFA correctly classified 86% of male unaffected relatives and 93% of male controls. In both sexes, upper facial width contributed most to group discrimination. Following DFA, unaffected relatives were assigned to risk/liability classes based on the degree of phenotypic divergence from controls. Results indicate that craniofacial shape differences characterizing unaffected relatives are partly sex-specific and are in broad agreement with previous reports. These findings further suggest that a quantitative assessment of the craniofacial phenotype may allow for the identification of susceptible individuals within nonsyndromic cleft families.     

Facial morphometrics in the identification of gene carriers of X-linked hypohidrotic ectodermal dysplasia

Roentgenographic measurements and morphometric analysis were employed in the investigation of contrasting patterns of craniofacial variation between normal individuals and those affected by X-linked hypohidrotic ectodermal dysplasia (HED). The research objective was to identify and describe the facial characteristics of heterozygous gene carriers who show minor expression of the disorder. In this study of 13 HED families with 16 affected males, 12 carriers, and 12 normal individuals, affected individuals had at least 3 of the following 4 clinical signs and symptoms: a) hypodontia, b) hypohidrosis, c) hypotrichosis, and d) clinically distinct facial physiognomy. By contrast, the gene carriers manifested only one or 2 or none of the 4 clinical manifestations. In a preliminary comparison of gene carriers vs. normal individuals, we have generated 2 discriminant functions (each based on 3 facial measurements taken either from the lateral or frontal cephalograms). These 2 functions correctly diagnose 100% of the gene carriers and normal HED relatives. Facial anomalies characteristic of the gene carriers were 1) abnormally narrow and short maxillary width and palatal depth dimensions; 2) very small and retrusive malar and maxillary regions; 3) markedly reduced lower facial depth, height and width dimensions; 4) small head height, prominent forehead, and high-set orbits; 5) a generalized, symmetric reduction of the whole craniofacial complex.     

Morphological variability in unrepaired bilateral clefts with and without cleft palate evaluated with geometric morphometrics

In subjects with orofacial clefts, there is an unresolved controversy on the effect of congenital maxillary growth deficiency vs. the effect of surgical intervention on the outcome of treatment. Intrinsic growth impairment in subjects with orofacial clefts can be studied by comparing facial morphology of subjects with untreated cleft and unaffected individuals of the same ethnic background. Bilateral cleft lip and palate is the most severe and least prevalent form of the orofacial cleft. The aim of this study was to compare facial morphology in subjects with unrepaired complete bilateral clefts and unaffected controls using geometric morphometrics. Lateral cephalograms of 39 Indonesian subjects with unrepaired bilateral complete cleft lip and alveolus (mean age: 24 years), or unrepaired bilateral complete cleft lip, alveolus, and palate (mean age: 20.6 years) and 50 age and ethnically matched controls without a cleft (25 males, 25 females, mean age: 21.2 years) were digitized and traced and shape variability was explored using principal component analysis, while differences between groups and genders were evaluated with canonical variate analysis. Individuals with clefts had a more pronounced premaxilla than controls. Principal component analysis showed that facial variation in subjects with clefts occurred in the anteroposterior direction, whereas in controls it was mostly in the vertical direction. Regression analysis with group, sex, and age as covariates and principal components from 1 to 6 as dependent variables demonstrated a very limited effect of the covariates on the facial shape variability (only 11.6% of the variability was explained by the model). Differences between cleft and non-cleft subjects in the direction of facial variability suggest that individuals with bilateral clefts can have an intrinsic growth impairment affecting facial morphology later in life.     

A homozygous 237-kb deletion at 1p31 identified as the locus for midline cleft of the upper and lower lip in a consanguineous family

Orofacial clefts are congenital defects that vary widely in type and severity, and can occur in isolation or in association with a variety of other defects. Herein, we describe a consanguineous family afflicted with a unique form of orofacial clefting manifesting as a facial midline defect that also involves mandibular and maxillary structures. All four affected sibs had median clefts of the upper and lower lips, tooth misalignment, and poor oral hygiene. Linkage analysis of 17 family members identified a 15.3-Mb pair recessive locus at 1p31 with a LOD score of 3.63. To the best of our knowledge, this is, to date, the first locus reported for facial midline clefting and the first recessive locus for an isolated orofacial defect. The locus harboured a novel intergenic deletion of 273 164 bp, for which all fully affected sibs were homozygous. We did not note any potentially pathogenic gene variant at the 1p31 locus via exome-sequencing analysis. The identified deletion could be harbouring a regulatory element for the gene associated with the orofacial defect. The best candidate for the putative target gene is LHX8, located 49 149 bp upstream of the deletion. The gene is known to be associated with facial development in several animals. Four other family members had a subclinical phenotype – a simple notch in the lower lip or an increase in the interdental distance between the lower incisors – indicative of very low-level expression of the trait.     

Genetic aspects of the BOR syndrome—branchial fistulas,ear pits,hearing loss,and renal anomalies

A pedigree of branchio-oto-renal dysplasia (the BOR syndrome) is reported, including the documentation by serial audiometric studies of the onset and rapid progression of hearing loss in the twin sister of an affected child. The literature on this syndrome is analyzed to derive some figures for use in genetic counseling of such families. Branchio-oto-renal dysplasia is an autosomal dominant disorder in which affected individuals may have preauricular pits, lachrymal duct stenosis, hearing loss, branchial fistulas or cysts, structural defects of the outer, middle, and inner ear, and renal anomalies, which may range from mild hypoplasia to complete absence. Not all features of the syndrome are expressed in all carriers of the gene, but few carriers lack all the features, and the pits, branchial clefts, and hearing loss, are frequently expressed. Those offspring of affected persons who have pits or fistulas are likely (about 80%) to have hearing loss of varying degrees of severity. A minority of heterozygotes (about 7%) may have hearing loss without pits or fistulas. The risk of severe renal malformation is probably fairly low. Whether families that show dominant inheritance of pits, clefts, and deafness without renal anomalies represent variants of the BOR syndrome or a separate entity (the BO syndrome), is still not clear. At present, any individual with preauricular pits and branchial clefts deserves both otologic and renal investigation.     

Genetic aspects of the BOR syndrome--branchial fistulas, ear pits, hearing loss, and renal anomalies.

A pedigree of branchio-oto-renal dysplasia (the BOR syndrome) is reported, including the documentation by serial audiometric studies of the onset and rapid progression of hearing loss in the twin sister of an affected child. The literature on this syndrome is analyzed to derive some figures for use in genetic counseling of such families. Branchio-oto-renal dysplasia is an autosomal dominant disorder in which affected individuals may have preauricular pits, lachrymal duct stenosis, hearing loss, branchial fistulas or cysts, structural defects of the outer, middle, and inner ear, and renal anomalies, which may range from mild hypoplasia to complete absence. Not all features of the syndrome are expressed in all carriers of the gene, but few carriers lack all the features, and the pits, branchial clefts, and hearing loss, are frequently expressed. Those offspring of affected persons who have pits or fistulas are likely (about 80%) to have hearing loss of varying degrees of severity. A minority of heterozygotes (about 7%) may have hearing loss without pits or fistulas. The risk of severe renal malformation is probably fairly low. Whether families that show dominant inheritance of pits, clefts, and deafness without renal anomalies represent variants of the BOR syndrome or a separate entity (the BO syndrome), is still not clear. At present, any individual with preauricular pits and branchial clefts deserves both otologic and renal investigation.     

EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies

Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially ‘atypical'' cases who often have complex genotype interactions). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis.     

Mutational analysis of the Sonic Hedgehog gene in 220 newborns with oral clefts in a South American (ECLAMC) population

Oral clefts generally have a multifactorial etiology. A number of genes contribute to the formation of the face and palate. Cleft lip and/or palate can occur in pedigrees with autosomal dominant holoprosencephaly due to mutations in Sonic Hedgehog (SHH). In addition, animal models have shown that SHH is involved in face development. We thus examined the human SHH gene in 220 newborn infants with nonsyndromic oral clefts registered by the Estudio Colaborativo Latinoamericano de Malformaciones Congenitas: ECLAMC (Latin American Collaborative Study of Congenital Malformations). We found 15 variant bands in 13 patients with oral clefts, representing five different base changes, all of which were found by sequencing to represent silent polymorphisms. Four occurred in introns. The alteration occurring in an exon, Ser190Ser, may create a consensus sequence for the 3'splice site 6 bp downstream of the original consensus sequence. Thus, we did not identify any clearly disease-causing mutation in SHH in these patients, and conclude that SHH mutations are not a frequent cause of isolated oral clefts in humans.     

Population screening and cascade testing for carriers of SMA

Spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseases, caused by absence of both copies of the survival motor neuron 1 (SMN1) gene. Identification of SMA carriers has important implications for individuals with a family history and the general population. SMA carriers are completely healthy and most are unaware of their carrier status until they have an affected child. A total of 422 individuals have been studied to identify SMA carriers. This cohort included 117 parents of children homozygously deleted for SMN1 (94% were carriers and 6% had two copies of SMN1; of these individuals, two in seven had the '2+0' genotype, two in seven were normal but had children carrying a de novo deletion and three in seven were unresolved), 158 individuals with a significant family history of SMA (47% had one copy, 49% had two copies and 4% had three copies of SMN1) and 146 individuals with no family history of SMA (90% had two copies, 2% had one copy and 8% had three copies of SMN1). The SMA carrier frequency in the Australian population appears to be 1/49 and the frequency of two-copy SMN1 alleles and de novo deletion mutations are both at least 1.7%. A multimodal approach involving quantitative analysis, linkage analysis and genetic risk assessment (GRA), facilitates the resolution of SMA carrier status in individuals with a family history as well as individuals of the general population, providing couples with better choices in their family planning.     

Cranio‐facial remodeling in domestic dogs is associated with changes in larynx position

The hyo‐laryngeal complex is a multi‐segmented structure integrating the oral and pharyngeal cavities and thus a variety of critical functions related to airway control, feeding, and vocal communication. Currently, we lack a complete understanding of how the hyoid complex, and the functions it mediates, can also be affected by changes in surrounding cranio‐facial dimensions. Here, we explore these relationships in a breed of domestic dog, the Portuguese Water Dog, which is characterized by strong cranio‐facial variation. We used radiographic images of the upper body and head of 55 adult males and 51 adult females to obtain detailed measures of cranio‐facial variation and hyoid anatomy. Principal components analysis revealed multiple orthogonal dimensions of cranio‐facial variation, some of which were associated with significant differences in larynx position: the larynx occupied a more descended position in individuals with shorter, broader faces than in those with longer, narrower faces. We then tested the possibility that caudal displacement of the larynx in brachycephalic individuals might reflect a degree of tongue crowding resulting from facial shortening and reduction of oral and pharyngeal spaces. A cadaver sample was used to obtain detailed measurements of constituent bones of the hyoid skeleton and of the tongue body, and their relationships to cranio‐facial size and shape and overall body size supported the tongue‐crowding hypothesis. Considering the presence of descended larynges in numerous mammalian taxa, our findings establish an important precedent for the possibility that laryngeal descent can be initiated, and even sustained, in part in response to remodeling of the face and cranium for selective pressures unrelated to vocal production. These integrated changes could also have been involved in hominin evolution, where the different laryngeal positions in modern humans compared with nonhuman primates have been traditionally linked to the evolution of speech but which are likely to be multifactorial.     

Anthropometric craniofacial pattern profiles in Down syndrome

A series of 21 anthropometric craniofacial measurements was performed on 199 individuals with Down syndrome (DS), age 6 months to 61 years. These were compared to age and sex-matched normal standards, and Z score pattern profiles were constructed. These profiles confirmed brachycephaly and reduced ear length. With increasing age, maxillary growth was reduced in comparison to mandibular growth. Clinically, this was manifested by a change in facial shape from the characteristic round face of infancy to an oval shape in later life. Stepwise forward discriminant function analysis identified a subset of three variables (ear length, maxillary arc, and upper facial depth) which could accurately classify greater than 99% of the individuals in the combined sample of affected and unaffected individuals. Of the subjects with DS, 96.8% were classified correctly. These findings demonstrate the usefulness of anthropometric craniofacial pattern profiles in defining abnormal facial dimensions in particular syndromes and documenting the changes that occur with age. The technique should facilitate syndrome recognition, identification of carriers, and comparisons between syndromes. © 1993 Wiley-Liss, Inc.     

Identifying psychophysiological risk for psychopathology: Examples from substance abuse and schizophrenia research

A problem confronting the search for psychopathology-related genes concerns the difficulty identifying gene carriers. Psychiatric diagnosis provides imperfect identification of affected individuals, and unaffected gene carriers go undetected. Psychophysiological measures may assist molecular genetic investigations by indicating genetic susceptibility for psychopathology, thus increasing the probability of identifying affected and unaffected gene carriers. Research strategies based on these premises are applied to the study of psychoactive substance use disorders and schizophrenia. Data are presented illustrating (1) that individual differences in inhibitory control involving autonomic and antisaccade eye movement measures and the P3 component of the event-related potential may be sensitive to susceptibility for substance use disorders, and (2) that eye tracking variables may identify genetic risk for schizophrenia.     

Mutation screening of IRF6 among families with non-syndromic oral clefts and identification of two novel variants: review of the literature

Non-syndromic oral clefts share the main clinical features of Van der Woude Syndrome (VWS), with the exception of the lower lip pit. Thus, about 15% of VWS cases are indistinguishable from cases with non-syndromic oral clefts. IRF6 mutations are the major cause of VWS; however, variants in this gene show strong association with non-syndromic oral clefts, with a higher increased risk among cases with cleft lip only (CLO). A total of 39 individuals, including 16 patients with CLO and 23 patients with a family history of cleft, were examined for IRF6 mutations in the present study. Seven variants, including five known (c.-75-4 A>; G, c.-73T>; C, c.459G>; T 5, c.820G>; A, and c.1060 + 37C>; T) and two novel (c.-75-23G>; C and c.1380G>; T), were found. Both novel variants were inherited from non-affected parents and we did not find also in the 120 control chromosomes. In silico analysis revealed that both c.1380G>; T and c.-75-23G>; C variants may disrupts a putative exonic splicing enhancer and intronic splicing binding site for SC35, respectively.Taken together, the presence of deleterious IRF6 variants in patients with non-syndromic oral clefts could be most likely an evidence for VWS. While, IRF6 variants could, at best, contribute to clefting as part of a complex inheritance pattern, with both additional genes and environmental factors having a role.     

Pseudodeficiency of arylsulfatase A: a counseling dilemma

Arylsulfatase A (ASA) deficiency is the cause of early and late onset metachromatic leukodystrophy (MLD). Low ASA levels are detected in some healthy individuals who are pseudodeficient (PD). PD individuals can be distinguished, because PD fibroblasts hydrolyze 14C-sulfatide at similar rates to normal fibroblasts. This has also been demonstrated in amniocytes and chorionic villi (CV). The genetic basis for PD is not clearly understood and is most likely heterogeneous with respect to allelic mutations of the ASA gene. It is hypothesized that the PD phenotype can either be due to PD/PD or PD/MLD genotypes, only the latter representing a potential risk to offspring. We report an unusual family where two siblings, both carriers of the classic late infantile MLD allele, are married to unrelated PD individuals. One couple has two PD offspring; their "at risk" status, due to the lack of an affected offspring is in question. The other couple terminated a fetus determined to be affected with a "MLD variant", most likely a compound heterozygote. Cautions prenatal counseling of PD families is essential. The population frequency of the PD phenotype is unknown.     

Functional analysis of natural mutations in two TWIST protein motifs

Autosomal-dominant hypercholesterolemia (ADH) has been identified as a major risk factor for coronary vascular disease (CVD) and is associated with mutations in the low-density lipoprotein receptor (LDLR) and the apolipoprotein B (APOB) gene. Since 1991 DNA samples from clinically diagnosed ADH patients have been routinely analyzed for the presence of LDLR and APOB gene mutations. As of 2001, 1,641 index patients (164 index patients per year) had been identified, while from 2001 onward a more sensitive, high-throughput system was used, resulting in the identification of 1,177 new index patients (average=294 index patients per year). Of these 1,177 index cases, 131 different causative genetic variants in the LDLR gene and six different causative mutations in the APOB gene were new for the Dutch population. Of these 131 mutations, 83 LDLR and four APOB gene mutations had not been reported before. The inclusion of all 2,818 index cases into the national screening program for familial hypercholesterolemia (FH) resulted in the identification of 7,079 relatives who carried a mutation that causes ADH. Screening of the LDLR and APOB genes in clinically diagnosed FH patients resulted in approximately 77% of the patients being identified as carriers of a causative mutation. The population of patients with ADH was divided into three genetically distinct groups: carriers of an LDLR mutation (FH), carriers of an APOB mutation (FDB), and non-LDLR/non-APOB patients (FH3). No differences were found with regard to untreated cholesterol levels, response to therapy, and onset of CVD. However, all groups were at an increased risk for CVD. Therefore, to ultimately identify all individuals with ADH, the identification of new genes and mutations in the genes that cause ADH is of crucial importance for the ongoing national program to identify patients with ADH by genetic cascade screening.     

Fabry disease in a large Nova Scotia kindred: carrier detection using leucocyte alpha-galactosidase activity and an NcoI polymorphism detected by an alpha-galactosidase cDNA clone.

Fabry disease is an X linked recessive disorder of glycosphingolipid metabolism resulting from a deficiency of the lysosomal hydrolase alpha-galactosidase (alpha-gal). Measurement of the enzyme activity, however, is not an accurate method for identification of female carriers among at risk relatives of affected males. The alpha-gal cDNA and gene have been cloned previously and found to provide useful probes for the molecular analysis of affected families but these clones have not been available to us. Thus, to analyse Fabry disease in Nova Scotia, especially within a large kindred known to contain 30 affected males and 50 possible carrier females, we isolated an independent cDNA for alpha-gal. Using this clone as a probe, the mutation in the Nova Scotia kindred was shown not to be a major DNA alteration, but was found to be linked to the rarer allele (frequency 0.20) of the polymorphic NcoI site located 3' to the gene. Affected males from two Nova Scotia families who cannot be associated with the kindred by history were also found to have the rarer NcoI allele, which suggests they are, in fact, part of the kindred. The coupling of the mutation to an infrequent marker also helped carrier identification in the kindred where all of 17 obligate carriers examined, including six who were not identified as carriers by enzyme assays, were found to be heterozygous for the RFLP. Thus, DNA analysis can be used for presymptomatic and prenatal diagnosis in most portions of the Nova Scotia kindred affected with Fabry disease.     

An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21

In our efforts to identify new loci responsible for non-syndromic autosomal recessive forms of deafness, DFNB loci, we have pursued the analysis of large consanguineous affected families living in geographically isolated areas. Here, we report on the study of a Lebanese family comprising nine members presenting with a pre-lingual severe to profound sensorineural isolated form of deafness. Linkage analysis led to the characterization of a new locus, DFNB21, which was assigned to chromosome 11q23-25. Already mapped to this chromosomal region was TECTA. This gene encodes alpha-tectorin, a 2155 amino acid protein which is a component of the tectorial membrane. This gene recently has been shown to be responsible for a dominant form of deafness, DFNA8/12. Sequence analysis of the TECTA gene in the DFNB21- affected family revealed a G to A transition in the donor splice site (GT) of intron 9, predicted to lead to a truncated protein of 971 amino acids. This establishes that alpha-tectorin mutations can be responsible for both dominant and recessive forms of deafness. Comparison of the phenotype of the DFNB21 heterozygous carriers with that of DFNA8/12-affected individuals supports the hypothesis that the TECTA mutations which cause the dominant form of deafness have a dominant-negative effect. The present results provide genetic evidence for alpha-tectorin forming homo- or heteromeric structures.      

The PPAR-gamma Pro12Ala polymorphism and risk of cognitive impairment in a longitudinal study

The Pro12Ala polymorphism in the PPAR-gamma gene has been associated with reduced incidence of type 2 diabetes. Although diabetes has been implicated as a risk factor for dementia, the association of Pro12Ala with cognitive impairment is unclear. Dementia and cognitive impairment without dementia (CIND) were determined during six annual follow-up evaluations in a cohort of 929 older Latinos. Among those with diabetes at baseline, there was an increased rate of dementia/CIND for Ala carriers compared to non-carriers (adjusted hazard ratio (HR)=2.5, 95% confidence interval (CI): 1.3-4.9) but not among non-diabetic participants (adjusted HR=0.94; 95% CI: 0.49-1.8). Among males, there was also an increased rate for Ala carriers (adjusted HR=2.7, 95% CI: 1.4-5.2) but not among female carriers (adjusted HR=0.88; 95% CI: 0.47-1.6). The rate of dementia/CIND was highest in diabetic male Ala carriers (adjusted HR=4.2; 95% CI: 1.5-11) compared to non-diabetic male carriers (adjusted HR=2.9; 95% CI: 1.1-7.4), diabetic female carriers (HR=1.6; 95% CI: 0.66-4.1), and non-diabetic female carriers (HR=0.52; 95% CI: 0.21-1.3). These data suggest that although the Ala variant is associated with a reduced risk of type 2 diabetes, it may increase the risk of cognitive impairment in individuals once diabetes has developed. Male Ala carriers may also have a greater risk of dementia/CIND.     

Genetic heterogeneity in Peutz-Jeghers syndrome

LKB1, the human gene encoding a serine threonine kinase, was recently identified as a susceptibility gene for Peutz-Jeghers syndrome (PJS), a disease characterized by the constellation of intestinal hamartomata, oral mucocutaneous hyperpigmentation, and an increased risk for gastrointestinal as well as extraintestinal malignancies. To date, the majority of individuals with PJS have been found to have genetic alterations in LKB1, most of which result in protein truncation. Additionally, linkage analyses have suggested a modicum of genetic heterogeneity, with the majority of PJS families showing linkage to the LKB1 locus. In this study, we evaluated five kindreds with greater than two affected family members, five PJS probands with only one other affected family member, as well as 23 individuals with sporadic PJS for mutations within the LKB1 gene. Conformation sensitive gel electrophoresis was utilized for the initial screen, followed by direct sequence analysis for characterization. Long-range PCR was used for the detection of larger genetic insertions or deletions. Mutation analysis revealed genetic alterations in LKB1 in two probands who had a family history of PJS. LKB1 mutations were detected in only four of the remaining 23 cases of sporadic PJS. These data suggest the presence of significant genetic heterogeneity for PJS and the involvement of other loci in this syndrome.