CHARGE and Kabuki Syndromes: Gene-Specific DNA Methylation Signatures Identify Epigenetic Mechanisms Linking These Clinically Overlapping Conditions.

Epigenetic dysregulation has emerged as a recurring mechanism in the etiology of neurodevelopmental disorders. Two such disorders, CHARGE and Kabuki syndromes, result from loss of function mutations in chromodomain helicase DNA-binding protein 7 (CHD7LOF) and lysine (K) methyltransferase 2D (KMT2DLOF), respectively. Although these two syndromes are clinically distinct, there is significant phenotypic overlap. We therefore expected that epigenetically driven developmental pathways regulated by CHD7 and KMT2D would overlap and that DNA methylation (DNAm) alterations downstream of the mutations in these genes would identify common target genes, elucidating a mechanistic link between these two conditions, as well as specific target genes for each disorder. Genome-wide DNAm profiles in individuals with CHARGE and Kabuki syndromes with CHD7LOF or KMT2DLOF identified distinct sets of DNAm differences in each of the disorders, which were used to generate two unique, highly specific and sensitive DNAm signatures. These DNAm signatures were able to differentiate pathogenic mutations in these two genes from controls and from each other. Analysis of the DNAm targets in each gene-specific signature identified both common gene targets, including homeobox A5 (HOXA5), which could account for some of the clinical overlap in CHARGE and Kabuki syndromes, as well as distinct gene targets. Our findings demonstrate how characterization of the epigenome can contribute to our understanding of disease pathophysiology for epigenetic disorders, paving the way for explorations of novel therapeutics.

A contiguous microdeletion syndrome at Xp23.13 with non-obstructive azoospermia and congenital cataracts.

Non-obstructive azoospermia accounts for 10-15% of male infertility, resulting in 60% of all cases of azoospermia and affecting about 1% of the male population. About 30% of these cases are due to Y chromosome microdeletions, chromosome abnormalities, or hormonal disorders. Pathogenic variants in genes on the sex chromosomes have key roles in spermatogenic failure. The co-occurrence of azoospermia and congenital cataracts ranges between 1 in 165,000 and 1 in 500,000. Our 28-year-old patient with normal intelligence and abnormally shaped teeth presented with both disorders. A microarray revealed a microdeletion at Xp23.13 with a whole NHS gene deletion as well as a contiguous deletion of two other genes [SCML1 and RAI2]. This observation represents the first report of non-obstructive azoospermia with congenital cataracts and a contiguous deletion of the SCML1 gene, a transcript of which is exclusively expressed in the testis. SCML1 is the putative culprit gene, which requires functional study or animal experiments. Our analysis of 60 known spermatogenesis failure-related genes by whole-exome sequencing revealed no other candidate. The Nance-Horan syndrome due to pathogenic variants in the NHS gene at Xp23.13 including whole gene deletion does not have azoospermia as a feature. Our report adds to the completeness of genetic counseling for an individual with azoospermia and congenital cataracts.

Identification of a dominant MYH11 causal variant in chronic intestinal pseudo-obstruction: Results of whole-exome sequencing.

Chronic Intestinal Pseudo-Obstruction (CIPO) is a rare gastrointestinal disorder, which affects the smooth muscle contractions of the gastrointestinal tract. Dominant mutations in the smooth muscle actin gene, ACTG2, accounts for 44%-50% of CIPO patients. Other recessive or X-linked genes, including MYLK, LMOD1, RAD21, MYH11, MYL9, and FLNA were reported in single cases. In this study, we used Whole-Exome Sequencing (WES) to study 23 independent CIPO families including one extended family with 13 affected members. A dominantly inherited rare mutation, c.5819delC (p.Pro1940HisfsTer91), in the smooth muscle myosin gene, MYH11, was found in the extended family, shared by 7 affected family members but not by 3 unaffected family members with available DNA, suggesting a high probability of genetic linkage. Gene burden analysis indicates that additional genes, COL4A1, FBLN1 and HK2, may be associated with the disease. This study expanded our understanding of CIPO etiology and provided additional genetic evidence to physicians and genetic counselors for CIPO diagnosis.

Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination.

BACKGROUND: The combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive. METHODS: We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model. RESULTS: Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis. CONCLUSIONS: The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease. (Funded by the National Institutes of Health and others.).

Biallelic mutations in huntington disease: A new case with just one affected parent, review of the literature and terminology.

Patients with biallelic mutations for Huntington disease (HD) are rare. We present a 46-year-old female with two expanded Huntingtin (HTT) alleles with just one known affected parent. This is the first reported patient with molecular studies performed to exclude HTT uniparental disomy (UPD). The proband had biparental inheritance of HTT alleles (42/44 CAG repeats). Given the negative UPD results, the proband's unaffected mother either had a reduced penetrance allele that expanded into the full mutation range during transmission to our patient or an unknown full HTT mutation and died before symptom onset, unlikely given no family history of HD and asymptomatic at age 59. We made the novel observation in our literature review that most patients with biallelic HD did not have two full HTT mutations. Most had one HTT allele that was in the intermediate or reduced penetrance ranges or 40 CAG repeats, the lowest limit of the full mutation range. Although the number of patients is small, when an allele in these size ranges was present, generally the age of HD onset was in the 50s. If the second HTT allele had >45 repeats, then onset was typically 20s-30s. While similar ages of onset have been reported for patients with one or two HTT mutations, patients with biallelic mutations may have later onset if an expanded HTT allele has ≤40 CAG repeats. Finally, we propose that "biallelic mutations" or "compound heterozygosity" are more accurate descriptive terms than "homozygosity" when there are two non-identical expanded HTT alleles.

A palindrome-mediated recurrent translocation with 3:1 meiotic nondisjunction: the t(8;22)(q24.13;q11.21).

Palindrome-mediated genomic instability has been associated with chromosomal translocations, including the recurrent t(11;22)(q23;q11). We report a syndrome characterized by extremity anomalies, mild dysmorphia, and intellectual impairment caused by 3:1 meiotic segregation of a previously unrecognized recurrent palindrome-mediated rearrangement, the t(8;22)(q24.13;q11.21). There are at least ten prior reports of this translocation, and nearly identical PATRR8 and PATRR22 breakpoints were validated in several of these published cases. PCR analysis of sperm DNA from healthy males indicates that the t(8;22) arises de novo during gametogenesis in some, but not all, individuals. Furthermore, demonstration that de novo PATRR8-to-PATRR11 translocations occur in sperm suggests that palindrome-mediated translocation is a universal mechanism producing chromosomal rearrangements.

TFAP2A mutations result in branchio-oculo-facial syndrome.

Branchio-oculo-facial syndrome (BOFS) is a rare autosomal-dominant cleft palate-craniofacial disorder with variable expressivity. The major features include cutaneous anomalies (cervical, infra- and/or supra-auricular defects, often with dermal thymus), ocular anomalies, characteristic facial appearance (malformed pinnae, oral clefts), and, less commonly, renal and ectodermal (dental and hair) anomalies. The molecular basis for this disorder is heretofore unknown. We detected a 3.2 Mb deletion by 500K SNP microarray in an affected mother and son with BOFS at chromosome 6p24.3. Candidate genes in this region were selected for sequencing on the basis of their expression patterns and involvement in developmental pathways associated with the clinical findings of BOFS. Four additional BOFS patients were found to have de novo missense mutations in the highly conserved exons 4 and 5 (basic region of the DNA binding domain) of the TFAP2A gene in the candidate deleted region. We conclude BOFS is caused by mutations involving TFAP2A. More patients need to be studied to determine possible genetic heterogeneity and to establish whether there are genotype-phenotype correlations.

Partial trisomy 8 mosaicism due to a pseudoisodicentric chromosome 8.

Chromosome 8 is the largest autosome in which mosaic trisomy is compatible with life. Constitutional trisomy 8 (T8) is estimated to occur in approximately 0.1% of all recognized pregnancies. The estimated frequency of trisomy 8 mosaicism (T8M), also known as Warkany syndrome, is about 1/25,000 to 50,000 liveborns, and is found to be more prevalent in males than females, 5:1. T8M is known to demonstrate extreme clinical variability affecting multiple systems including central nervous, ocular, cardiac, gastrointestinal, genitourinary, and musculoskeletal. There appears to be little correlation between the level of mosaicism and the extent of the clinical phenotype. Additionally, the exact mechanism that causes the severity of phenotype in patients with T8M remains unknown. We report on a mildly dysmorphic male patient with partial low-level T8M due to a pseudoisodicentric chromosome 8 with normal 6.0 SNP microarray and high resolution chromosome analyses in lymphocytes. The aneuploidy was detected in fibroblasts and confirmed by FISH in lymphocytes. This report elaborates further the clinical variability seen in trisomy 8 mosaicism.

Genotype-phenotype analysis of the branchio-oculo-facial syndrome.

Branchio-oculo-facial syndrome (BOFS; OMIM#113620) is a rare autosomal dominant craniofacial disorder with variable expression. Major features include cutaneous and ocular abnormalities, characteristic facies, renal, ectodermal, and temporal bone anomalies. Having determined that mutations involving TFAP2A result in BOFS, we studied a total of 30 families (41 affected individuals); 26/30 (87%) fulfilled our cardinal diagnostic criteria. The original family with the 3.2 Mb deletion including the TFAP2A gene remains the only BOFS family without the typical CL/P and the only family with a deletion. We have identified a hotspot region in the highly conserved exons 4 and 5 of TFAP2A that harbors missense mutations in 27/30 (90%) families. Several of these mutations are recurrent. Mosaicism was detected in one family. To date, genetic heterogeneity has not been observed. Although the cardinal criteria for BOFS have been based on the presence of each of the core defects, an affected family member or thymic remnant, we documented TFAP2A mutations in three (10%) probands in our series without a classic cervical cutaneous defect or ectopic thymus. Temporal bone anomalies were identified in 3/5 patients investigated. The occurrence of CL/P, premature graying, coloboma, heterochromia irides, and ectopic thymus, are evidence for BOFS as a neurocristopathy. Intrafamilial clinical variability can be marked. Although there does not appear to be mutation-specific genotype-phenotype correlations at this time, more patients need to be studied. Clinical testing for TFAP2A mutations is now available and will assist geneticists in confirming the typical cases or excluding the diagnosis in atypical cases.

A neuroligin-4 missense mutation associated with autism impairs neuroligin-4 folding and endoplasmic reticulum export.

Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4) (gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations and the limited information about the affected patients and the functional consequences of their mutations cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino-acid substitution in NL4 (R87W). This substitution was absent from the brothers' asymptomatic parents, suggesting that it arose in the maternal germ line. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell surface. As a result, the R87W substitution inactivated the synapse-formation activity of NL4 and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism.

Mosaic Down syndrome in a patient with low-level mosaicism detected by microarray.

Down syndrome (DS) is the most common aneuploidy in liveborns with an estimated frequency of 1 in 650-1,000 births. Approximately 1-2% of all live-born DS individuals have mosaicism. The correlation between the percentage of mosaicism and the severity of the phenotype in mosaic trisomy 21 has been determined in previous studies. Patients with low percent of trisomy 21 have less phenotypic manifestations, higher IQs, and better overall survival. We report on a 1-day-old baby girl with subtle features of DS and low-level trisomy 21 mosaicism (8-13% in lymphocytes, 31% in buccal cells) with normal high resolution chromosome analysis. The aneuploidy was detected by 6.0 SNP microarray and confirmed by fluorescent in situ hybridization (FISH). Further studies to detect mosaicism are recommended from blood (using interphase FISH) or other tissues in the evaluation of a child with features of DS and a normal blood metaphase karyotype. SNP microarray technology appears to be a useful adjunct, being able to detect low-level mosaicism in these cases.

Novel de novo PCDH19 mutations in three unrelated females with epilepsy female restricted mental retardation syndrome.

Epilepsy and Mental Retardation Limited to Females (EFMR) [OMIM 300088] was first described in 1971 [Juberg and Hellman, 1971] in 15 related females with early onset grand mal seizures and mental retardation. Although EFMR demonstrates X-linked inheritance, it follows an unusual pattern by sparing transmitting males and affecting only heterozygous females. In 2008, mutations within the protocadherin 19 (PCDH19) gene were implicated as causative of EFMR [Dibbens et al. (2008); Nat Genet 40:776-781]. The EFMR phenotype is typically characterized by seizure onset in infancy and mild to severe intellectual impairment. Several individuals with EFMR have also been described as having autistic features. We describe three unrelated female individuals, ranging in age from 3 to 19 years, with de novo novel PCDH19 mutations. All three individuals have seizure onset in infancy and require the use of multiple antiepileptic drugs. They also have varying degrees of intellectual impairment along with the presence of autistic features. Although most individuals with EFMR described to date demonstrate this unusual familial X-linked inheritance, our three unrelated females with de novo mutations highlight the importance of testing PCDH19 in females with early onset epilepsy, intellectual impairment, and autistic features, regardless of family history.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Prenatal molecular diagnosis of tuberous sclerosis complex.

OBJECTIVE: The objective of the study was to report experience with prenatal molecular diagnosis of tuberous sclerosis complex (TSC). STUDY DESIGN: Sequential deoxyribonucleic acid (DNA) studies were performed on amniotic fluid cells and chorionic villi from 50 pregnant women at risk for having a child with TSC. Mutations were determined by gene sequencing and deletion/duplication analysis of the 2 TSC genes. RESULTS: DNA analysis was successful in 48 of 50 tested fetuses. Mutations were precisely identified in a family member (24) (TSC1 [5]; TSC2 [19]) and/or fetus (11) (TSC1 [3]; TSC2 [8]). Novel mutations were found in 19 individual families, and a probable polymorphism was noted in 4. Second-trimester ultrasound detected 18 fetuses with cardiac rhabdomyomas. There was insufficient DNA in 1, whereas 8 of 17 (47%) had a mutation, 6 (75%) being in TSC2. In 4 of 18 cases, a mutation was detected in the fetus for the first time despite a parent known to have TSC. CONCLUSION: The value and utility of prenatal diagnosis of TSC by DNA analysis was demonstrated by the results in this series of 50 pregnancies in women at risk of having affected offspring. A family history of TSC or detection of fetal cardiac rhabdomyoma should prompt genetic evaluation and counseling of parents and the option of prenatal diagnosis.

Neuroimaging findings in macrocephaly-capillary malformation: a longitudinal study of 17 patients.

Here, we report the neuroimaging findings and neurological changes in 17 unpublished patients with Macrocephaly-Capillary Malformation (M-CM). This syndrome has been traditionally known as Macrocephaly-Cutis Marmorata Telangiectatica Congenita (M-CMTC), but we explain why M-CM is a more accurate term for this overgrowth syndrome. We analyzed the 17 patients with available brain MRI or CT scans and compared their findings with features identified by a comprehensive review of published cases. White matter irregularities with increased signal on T2-weighted images were commonly observed findings. A distinctive feature in more than half the patients was cerebellar tonsillar herniation associated with rapid brain growth and progressive crowding of the posterior fossa during infancy. In four such cases, we confirmed that the tonsillar herniation was an acquired event. Concurrently, with the development of these findings, ventriculomegaly (frequently obstructive) and dilated dural venous sinuses were observed in conjunction with prominent Virchow-Robin spaces in many of those in whom cerebellar tonsil herniation had developed. We postulate that this constellation of unusual features suggests a dynamic process of mechanical compromise in the posterior fossa, perhaps initiated by a rapidly growing cerebellum, which leads to congestion of the venous drainage with subsequently compromised cerebrospinal fluid reabsorption, all of which increases the posterior fossa pressure and leads to acquired tonsillar herniation. We make a distinction between congenital Chiari I malformation and acquired cerebellar tonsil herniation in this syndrome. We also observed numerous examples of abnormal cortical morphogenesis, including focal cortical dysplasia, polymicrogyria which primarily involved the perisylvian and insular regions, and cerebral and/or cerebellar asymmetric overgrowth. Other findings included a high frequency of cavum septum pellucidum or vergae, thickened corpus callosum, prominent optic nerve sheaths and a single case of venous sinus thrombosis. One patient was found to have a frontal perifalcine mass resembling a meningioma at age 5 years. This is the second apparent occurrence of this specific tumor in M-CM.

Dental issues in lacrimo-auriculo-dento-digital syndrome: An autosomal dominant condition with clinical and genetic variability.

BACKGROUND AND OVERVIEW: Lacrimo-auriculo-dento-digital (LADD) syndrome is an autosomal dominant disorder with variable lacrimal and salivary gland hypoplasia and aplasia, auricular anomalies and hearing loss, dental defects and caries, and digital anomalies. CASE DESCRIPTION: The authors present the cases of 2 unrelated children with enamel defects and history of dry mouth leading to recurrent dental caries. The referring diagnoses were Sjögren disease and hypohidrotic ectodermal dysplasia, respectively. The geneticist suspected LADD syndrome, which was confirmed by means of molecular studies showing mutations of 2 genes: fibroblast growth factor receptor 2 and fibroblast growth factor 10, respectively. Similarly affected relatives indicated an autosomal dominant inheritance. These relatives needed multiple dental rehabilitations during childhood and dentures in adulthood. CONCLUSIONS AND PRACTICAL IMPLICATIONS: Dry mouth, multiple caries, enamel defects, and abnormal tooth morphology were the reasons for seeking care from dentists. However, clinical evaluation and diagnostic imaging studies helped identify anomalies of the lacrimal and salivary glands, ears, and digits, indicating involvement of different areas of the body, compatible with LADD syndrome. Accordingly, dentists should consider genetic disorders in patients with multiple anomalies. For instance, oculodentodigital syndrome, oral-facial-digital syndrome, and LADD syndrome (among others) may have dental issues as the major clinical manifestation. Accurate identification of a particular syndrome is now commonplace with the use of genetic testing. When a patient has multiple anomalies suggestive of a syndromic condition, appropriate genetic testing can help verify the clinical diagnosis. Keeping genetics in mind helps earlier identification of other affected family members with diagnostic genetic testing and appropriate treatment; the economic advantage is to shorten the diagnostic odyssey and possibly preserve dentition.

Prenatal and postnatal characterization of a de novo Xq22.1 terminal deletion.

We present a case of a de novo Xq22.1 chromosomal terminal deletion discovered prenatally by conventional cytogenetics. The pregnancy resulted in the birth of a normal girl. Preferential inactivation of the abnormal X was demonstrated postnatally. Fluorescence in situ hybridization (FISH) demonstrated a terminal Xq deletion spanning Xq22.1 -->qter. An X painting probe ruled out a translocation. The deleted X chromosome was determined to be of paternal origin. The girl is now 4 years old with normal physical and psychomotor development. X chromosomal deletions are infrequent findings in prenatal diagnosis and present a difficult counseling challenge when they occur. Prenatal X-inactivation studies provide an opportunity for more informative genetic counseling when a de novo X chromosome deletion is detected.

The Genetics of Benign Paroxysmal Torticollis of Infancy: Is There an Association With Mutations in the CACNA1A Gene?

Benign paroxysmal torticollis of infancy is an unusual movement disorder, often accompanied by a family history of migraine. Some benign paroxysmal torticollis cases are associated with CACNA1A mutations. The authors sought to determine the frequency of CACNA1A mutations in benign paroxysmal torticollis by testing 8 children and their parents and by searching the literature for benign paroxysmal torticollis cases with accompanying CACNA1A mutations or other disorders linked to the same gene. In our 8 benign paroxysmal torticollis cases, the authors found 3 different polymorphisms, but no pathogenic mutations. By contrast, in the literature, the authors found 4 benign paroxysmal torticollis cases with CACNA1A mutations, 3 with accompanying family histories of 1 or more of familial hemiplegic migraine, episodic ataxia, and paroxysmal tonic upgaze. Thus, CACNA1A mutations are more likely to be found in children with benign paroxysmal torticollis if accompanied by family histories of familial hemiplegic migraine, episodic ataxia, or paroxysmal tonic upgaze.