Thomas A. Maher

LADD syndrome is caused by FGF10 mutations

Lacrimo‐auriculo‐dento‐digital syndrome [LADD (MIM 149730)] is an autosomal‐dominant multiple congenital anomaly disorder characterized by aplasia, atresia or hypoplasia of the lacrimal and salivary systems, cup‐shaped ears, hearing loss, and dental and digital anomalies. Loss of function mutations in FGF10 were recently described in aplasia of the lacrimal and salivary glands [ALSG (MIM 180920; MIM 103420)] (Entesarian et al., Nat Genet 2005: 37: 125–127, Milunsky et al., American College of Medical Genetics Annual Meeting, Dallas, TX, 2005: A100). Due to the significant phenotypic overlap between LADD syndrome and ALSG and the variable expressivity of both the disorders, we hypothesized that FGF10 mutations could also result in LADD syndrome. A de novo missense mutation was found in exon 3 of FGF10 in a 3‐year‐old female (Family 1) with LADD syndrome. This missense mutation, resulting in a non‐conservative amino acid change, was confirmed by restriction enzyme digestion and was not found in 500 control chromosomes. A nonsense mutation was also found in exon 2 of FGF10 (Family 2) in a 19‐year‐old mother with ALSG and her 2‐year‐old daughter with LADD syndrome. Previous studies of FGF10 mutant mice have demonstrated abnormalities consistent with ALSG and LADD syndrome. We conclude that ALSG and LADD syndrome may represent variable presentations of the same clinical spectrum caused by FGF10 mutations.

Molecular, biochemical, and phenotypic analysis of a hemizygous male with a severe atypical phenotype for X-linked dominant conradi-hunermann-happle syndrome and a mutation in EBP

X‐linked dominant Conradi‐Hunermann‐Happle syndrome (CDPX2; MIM 302960) is a rare chondrodysplasia punctata primarily affecting females. CDPX2 is presumed lethal in males, although a few affected males have been reported. CDPX2 is a cholesterol biosynthetic disorder due to 3‐β‐hydroxysteroid‐Δ8,Δ7‐isomerase deficiency caused by mutations in the emopamil binding protein (EBP) gene. A 2.5‐year‐old Caucasian male was followed from the age of 6 weeks and noted to have significant developmental delay, hypotonia, seizures, and patchy hypopigmentation. Multiple congenital anomalies included a unilateral cataract, esotropia, crossed renal ectopia, stenotic ear canals, and failure to thrive, requiring G‐tube placement. Multiple minor anomalies and ptosis were noted. No skeletal asymmetry or chondrodysplasia punctata were noted on skeletal survey at 6 weeks and 13 months. An extensive genetic work‐up including cholesterol (126–176 mg/dl) and 7‐dehydrocholesterol was unrevealing. However, the levels of 8(9)‐cholestenol and 8‐dehydrocholesterol were mildly increased in plasma, which was confirmed in cultured fibroblasts. This prompted molecular analysis of the EBP gene, which revealed a novel hemizygous (nonmosaic) mutation in exon 2 (L18P). Two restriction digests were developed that confirmed this mutation in skin fibroblasts, blood, and buccal cells (all nonmosaic). We determined that the patients mother (adopted) also has the L18P mutation enabling prenatal diagnosis of a normal male fetus. She has normal stature, no asymmetry, no cataracts at this time, and has a patch of hyperpigmentation on her chest best visualized on Woods lamp examination, characteristic of CDPX2. The mild maternal phenotype has been described previously. However, this nonmosaic missense mutation has resulted in a severe phenotype in her surviving son.

Fragile X carrier screening and spinocerebellar ataxia in older males

The report by Jacquemont et al. [2003] raises the important question of whether to perform fragile X carrier testing on those males presenting after age 50 with spinocerebellar ataxia (SCA) symptomatology. A newly described disorder that includes intention tremor, cerebellar ataxia, Parkinsonism, and generalized brain atrophy has been reported in a subset of fragile X carrier males older than age 50 [Hagerman et al., 2001; Leehey et al., 2002]. A major radiographic criterion of this new disorder named fragile X–associated tremor/ataxia syndrome (FXTAS) is hyperintensity on the T2 weighted magnetic resonance images in the cerebellar white matter and middle cerebellar peduncles [Brunberg et al., 2002]. Molecular findings include elevated mRNA and low-normal or mildly decreased levels of fragile X mental retardation 1 protein. Ithasbeenestimatedthat1 in813 males [Dombrowski et al., 2002] has a fragile X premutation (55–200 CGG repeats). In an attempt to ascertain the prevalence of FXTAS in a submitted male cohort greater than age 50 with SCA symptomatology, we retrospectively and anonymously performed the following study. A total of 167 male patients greater than age 50 years submitted for SCA testing and found to be negative for the SCA genes 1,2,3,6,7,8,10,12, and DRPLA, were amplified for the FMR1 FRAXA locus using standard procedures. One male greater than age 50 years with a FMR1 expansion of 80 repeats was detected in this cohort. As these patients were submitted to our laboratory for SCA testing, we do not have data on family history, MRI findings, or specific symptomatology. Hence, this cohort does not represent rigorous clinical analysis, but it is believed that all have gait ataxia. Chi square analysis did not yield statistical significance. Hence, finding one individual from our cohort who is a premutation carrier may be a chance finding. Further studies are warranted, but it appears that the prevalence of FXTAS in the population studied is low.

Connexin-26 Gene Analysis in Hearing-Impaired Newborns

The efficacy and utility of the Connexin-26 (Cx-26) gene (also called GJB2) analysis from DNA isolated from Guthrie newborn screening cards is demonstrated. This analysis precisely defined a major cause of prelingual nonsyndromic deafness in those children requiring amplification in our study. Guthrie cards were obtained from 49 deaf children requiring amplification identified over the last 5 years by the Rhode Island Newborn Screening Program. Children with syndromes or other recognizable causes of hearing loss were excluded. DNA was extracted from the Guthrie cards and analyzed sequentially for the Cx-26 35delG mutation and then for the 167delT mutation followed by gene sequencing on remaining heterozygotes. Three of 42 children were 35delG homozygotes; 2/42 children were 35delG/167delT compound heterozygotes. One child was identified as being a 35delG heterozygote with no other mutation found by sequencing. Nine Guthrie cards yielded no amplification or uninterpretable results. Cx-26 mutations were identified as causing 11.9% of the deafness in the children studied. In conclusion, Cx-26 analysis is an important test that identifies a major cause of prelingual nonsyndromic deafness. Molecular analysis of hearing-impaired newborns will be important for genetic counseling in these families. Failures with Guthrie cards may make use of other collection methods preferable.

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.

Mutation Analysis in Rett Syndrome

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome. We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome patients to characterize the genotype-phenotype spectrum. Bidirectional sequencing of the entire MECP2 coding region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had been reported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified mutations have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified mutations in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop acquired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate the need for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common mutations followed by sequencing, when necessary. Analysis of common mutations before sequencing the entire gene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.

A re-examination of the chromosome 8p22-8p23.1 region in Kabuki syndrome

To the Editor: Milunsky and Huang reported six Kabuki syndrome (KS) patients studied bymetaphase comparative genomic hybridization (CGH) andmetaphase bacterial artificial chromosome-fluorescent in situ hybridization (BAC-FISH) with what was interpreted as a chromosome 8p22-8p23.1 duplication (1). Also reported was a paracentric inversion in the patients and two of the unaffected mothers. Multiple studies have failed to confirmaduplication of this region inKSusingBAC-FISHorarrayCGH (2–10). A triplication of the region has now been reported in a patient with features overlapping KS (11). A critical re-examination of our data with additional molecular experiments and a blinded repetition of the original six reported KS patient BAC-FISH data with controls in metaphase and this time in interphase was performed. It was also discovered that in Figure 2 of the original manuscript, 2 images of 30 submitted (2c/6c and 3d/5d) were unintentionally identical (1). The authors apologize for this error. We first sought molecular evidence of a microduplication involving chromosome 8p22-8p23.1 using various molecular techniques. We used the ABI 3100 sequencer to compare multiple dinucleotide markers across the region in affected patients vs controls. We found only two alleles in our affected patients with equal intensities. We then employed matrix-assisted laser desorption– ionization time-of-flight mass spectrometry for single nucleotide polymorphism (SNP) analysis and did not find evidence of a duplication in this region. We then examined GATA4 and CTSB as possible candidate genes by multiplex ligationdependent probe amplification and did not detect either a duplication or a deletion. Finally, we interrogated our samples using a 500K Affymetrix SNP microarray that did not demonstrate any copy number changes in the 8p22-8p23.1 region. Finding no duplication in this region by multiple molecular methods, we then proceeded to sequence all the coding exons and exon–intron boundaries in four genes within the region (GATA4, NEIL2, FDFT1, and CTSB). Although several known polymorphisms were found at a similar frequency to our normal control DNA samples, we found no pathogenic mutations in any of the KS patients studied. We then re-examined the region cytogenetically. Interphase FISH analyses were performed using the probes located at the 8p duplicated’ region and a centromere probe of chromosome 8 as a control. One thousand interphase cells per patient were analyzed, and the FISH results were all below the normal duplication cutoff, which supports no evidence of an 8p2 duplication in the KS patients studied. A paracentric inversion was originally reported in the KS patients and two mothers using the RP11-122N11 probe. We now recognize that this probe has a genomic segmental duplication and was misinterpreted to be an inversion because of the cross hybridization of the probe between the target region and its surrounding region 400 kb away. Hence, there is no evidence of a paracentric inversion. Our reported 8p segmental duplication was based on a different signal pattern between homologues by metaphase FISH in the KS patients. We again demonstrated this difference between chromosome 8 homologues. Knowing now that there is no duplication underlying this difference, further investigations to explain this phenomenon are in progress, including quantitative twodimensional and three-dimensional analyses of the FISH signals. Although our original data were misinterpreted, the repetition of the experiment again showing a difference between the chromosome 8 homologues may still signal a clue that may help identify the underlying etiology of KS.

Pallister–Killian syndrome: tetrasomy of 12pter→12p11.22 in a boy with an analphoid, inverted duplicated marker chromosome

Supernumerary marker chromosomes (SMCs) without detectable alphoid DNA are predicted to have a neocentromere and have been referred to as mitotically stable neocentromere marker chromosomes (NMCs). Here we report the molecular cytogenetic characterization of a new case of Pallister–Killian syndrome (PKS) in a boy with an analphoid, inverted duplicated NMC derived from 12pter→12p11.22 in his fibroblasts by using high‐resolution comparative genetic hybridization (HR‐CGH), multiplex fluorescent in situ hybridization (FISH) and bacterial artificial chromosome (BAC)‐FISH mapping analyses with various alpha‐satellite DNA probes, subtelomere probes and BAC‐DNA probes. Precise identification of SMCs and NMCs is of essential importance in genetic counseling. HR‐CGH is a more informative and often a faster way of precisely identifying the origin of SMCs. This case is the third report of PKS with an NMC containing an inverted duplication of partial 12p with available clinical data. These observations may help to determine the critical region for PKS and the mechanisms leading to the origin of the NMC derived from 12pter→12p11.22 – a region that appears to be susceptible to the formation of neocentromeres. The use of subtelomeric probe PCP12p in buccal cells appears superior to the use of the centromere probe D12Z3 for the diagnosis of the PKS.

Fraternal twins with Aarskog-Scott syndrome due to maternal germline mosaicism

Aarskog–Scott syndrome is a rare X‐linked recessive disorder with characteristic facial, skeletal, and genital abnormalities. We report on Aarskog–Scott syndrome in male dizygotic twins with an identical de novo mutation in FGD1 that resulted from germline mosaicism in the phenotypically normal mother. This is the first report of inheritance by germline mosaicism for the FGD1 gene.

Prenatal diagnosis of myotonic muscular dystrophy with linked deoxyribonucleic acid probes

Since the localization of the myotonic muscular dystrophy gene, closer deoxyribonucleic acid markers have been discovered. These now facilitate both presymptomatic and prenatal diagnosis of myotonic muscular dystrophy. We report our prenatal diagnosis experience with six cases in five families. Obstetricians are advised to inform their patients with a family history of myotonic muscular dystrophy of these testing opportunities. The fetus of the mother with myotonic muscular dystrophy who remains in utero until term is at considerable risk, as is the mother herself, of serious obstetric complications.