No abstract

The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260 kb 3' of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.

Mutations in the EGR2 gene cause a spectrum of Charcot-Marie-Tooth disease and related inherited peripheral neuropathies. We ascertained ten consecutive patients with various EGR2 mutations, report a novel de novo mutation, and provide longitudinal clinical data to characterize the natural history of the peripheral neuropathy. We confirmed that respiratory compromise and cranial nerve dysfunction are commonly associated with EGR2 mutations and can be useful in guiding molecular diagnosis. We also contrast morphological studies in the context of the I268N homozygous recessive mutation affecting the NAB repressor binding site and the R359W dominant-negative mutation in the zinc-finger domain.

Heterozygous mutations in the early growth response gene 2 (EGR2), which encodes a zinc-finger transcription factor that regulates the late stages of myelination, cause myelinopathies including congenital hypomyelinating neuropathy, Dejerine-Sottas neuropathy (DSN), and Charcot-Marie-Tooth disease type 1. We screened 170 unrelated neuropathy patients without mutations involving the peripheral myelin protein 22 gene (PMP22), the myelin protein zero gene (MPZ), or the gap junction protein beta1 gene (GJB1) and identified two DSN patients with the heterozygous mutation R359W in the alpha-helix domain of the first zinc-finger of EGR2. We now report that this mutation is a recurrent cause of DSN, and that expressivity ranges from that typical for DSN to a more rapidly progressive neuropathy that can cause death by age 6 years. Furthermore, in contrast to patients with typical DSN, patients with the EGR2 R359W mutation have more respiratory compromise and cranial nerve involvement.

Point mutations in the zone of polarizing activity regulatory sequence (ZRS) are known to cause human limb malformations. Although most mutations cause preaxial polydactyly (PPD), triphalangeal thumb (TPT) or both, a mutation in position 404 of the ZRS causes more severe Werner mesomelic syndrome (WMS) for which malformations include the distal arm or leg bones in addition to the hands and/or feet. Of more than 15 reported families with ZRS mutations, only one homozygous individual has been reported, with no change in phenotype compared with heterozygotes. Here, we describe a novel point mutation in the ZRS, 402C>T (AC007097.4:g.105548C>T), that is transmitted through two Mexican families with one homozygous individual. The homozygous phenotype for this mutation, WMS, is more severe than the numerous heterozygous individuals genotyped from both families who have TPT and PPD. A mouse transgenic enhancer assay shows that this mutation causes an expansion of the enhancer’s expression domain in the developing mouse limb, confirming its pathogenicity. Combined, our results identify a novel ZRS mutation in the Mexican population, 402C>T, and suggest that a dosage effect exists for this ZRS mutation.

The results suggest that approximately 9.2% of patients in the heterogeneous population with an LGMD diagnosis will show mutations of the calpain III gene. Interestingly, two patients were heterozygous for a single mutation at the DNA level, whereas only the mutant allele was observed at the RNA level. This suggests that there are undetectable, nondeletion mutations that ablate expression of the calpain III gene.

Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS-CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non-CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS-CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14 000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high-density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high-density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions. G enetic disorders can be caused by an enormously broad range of genomic aberrations. These range from singlenucleotide point mutations to duplications and deletions of millions of basepairs of DNA, up to entire chromosomes, as in Down's syndrome. An ongoing challenge for genetic diagnostics is to develop methods to sensitively and specifically diagnose this entire range of mutations. Although current methods reliably detect the largest and smallest mutations (microscopic cytogenetics and sequencing, respectively), the middle range of mutations is difficult to detect. We dealt with this challenge by applying the evolving technology of arraybased comparative genomic hybridisation (CGH) to a disorder that is caused by a wide range of mutations, including mutations in this intermediate range.Haploinsufficiency of the transcription factor gene GLI3, on chromosome 7p14.1, causes Greig cephalopolysyndactyly syndrome (GCPS).1 Many mutations have been identified, including translocations and large genomic deletions, as well as small intragenic deletions or duplications 2 and point mutations.2 3 This wide range of mutations complicates molecular diagnostics, requiring numerous tests to detect all possible mutations. In addition, when...

Case Report K.R., a black female, is the first child of a 22-year-old mother and 21-year-old father. She weighed 3360 g at birth and was born at term by a spontaneous vaginal delivery. There was no family history of congenital malformations or mental retardation, and the parents were not related. No drugs were ingested during the early part of pregnancy and there were no prenatal infections. However, the gestation was complicated by peripheral oedema in the eighth month treated with a two-week course of diuretics. The patient was referred to the University of California Medical Center at 4 weeks of age for failure to thrive, recurrent regurgitation, and tachypnoea without cyanosis while feeding.Positive physical findings included a prominent occiput, superiorly pointed (elfin) ears with incomplete helices, a shortened nasal septum causing an upturned nose, a midline cleft of the palate, a bifid uvula, mild micrognathia, poor oropharyngeal muscular tone in contrast to good generalized tone, a grade III/VI harsh systolic murmur, an accentuated pulmonic second heart sound, an enlarged liver, a 1 cm umbilical hernia, a 1 cm caf&-au-lait spot in the right lower quadrant of the abdomen, a sacral dimple, a short distal phalanx of the left fifth finger with an absent extensor crease, and bilateral plantar displacement of the third toes (see Fig. 1).Dermatoglyphics revealed seven ulnar loops, two whorls, and one radial loop with both palms having a single axial triradius (atd angle= 25°). A hallucal arch pattern was present bilaterally.An intravenous pyelogram showed a duplicated left intrarenal collecting system. Cardiac catheterization with angiograms revealed pulmonary hypertension, an enlarged right atrium and ventricle, a small atrial septal defect (ASD) with left-to-right shunting, and inadequate alveolar ventilation. No cause for the last finding other than the relative macroglossia, increased pharyngeal secretions, and poor oropharynx muscle control was found. For the same reasons poor feeding was a continuing problem.An endotracheal tube improved ventilation and at 6 -4 AJ Hunter, H. (196Q). A controlled study of the psychopathology and physical measurements of Klinefelter's syndrome.