G. Neri

Characterization of MLH1 and MSH2 alternative splicing and its relevance to molecular testing of colorectal cancer susceptibility

Abstract The phenomenon of alternative splicing in the DNA mismatch repair genes MLH1 and MSH2 was extensively investigated by coupled reverse transcription-polymerase chain reaction in different human tissues, including 42 mononuclear blood cell samples – 31 obtained from familial colon cancer patients or their at-risk relatives and 11 from healthy blood donors – 7 normal colonic mucosae, 4 established human cancer cell lines, 8 colorectal tumors, and one sample each of ileum, liver, muscle, thymus, breast, and EBV-transformed lymphoblasts. Several isoforms were observed for each gene. Products of MLH1 alternative splicing included mRNAs lacking alternative exons 6/9, 9, 9/10, 9/10/11, 10/11, 12, 16, and 17. For MSH2, products lacking exons 5, 13, 2 through 7, and 2 through 8 were identified. The levels of expression were found to vary among different samples. All isoforms were found in a relevant fraction (43–100%) of the mononuclear blood cell samples, as well as in other tissues. The splicing variants were also detected in normal colonic mucosa, with the exceptions of the MLH1–6/9 and –10/11 and the MSH2–13 isoforms. Germline mutations of MLH1 and MSH2 confer constitutional predisposition to the development of colorectal cancer and other neoplasms. A substantial proportion of the mutations identified so far involve alterations of the normal splicing process. Knowledge of the existence of multiple alternative splicing events, not caused by genomic DNA changes, is important for the evaluation of the results of molecular diagnostic tests based on RNA analysis.

Hirschsprung disease, mental retardation, characteristic facial features, and mutation in the gene ZFHX1B (SIP1): Confirmation of the Mowat-Wilson syndrome

We read with interest the article ‘‘Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22–q23’’ [Mowat et al., 1998]. The same distinctive face was reported by Kääriäinen et al. [2001] in four out of five patients in our opinion and by Zweier et al. [2002]. Recently, mutations in the zinc finger homeobox 1 B gene, ZFHX1B (SIP1), were identified in patients with and without Hirschsprung disease (HSCR), multiple congenital anomalies, mental retardation (MCA/MR), and the same facial gestalt [Zweier et al., 2002]. The latter authors propose the name, Mowat-Wilson syndrome for this entity. It has been demonstrated that patients 1, 2, and 3 of Mowat et al. [1998] also have a heterozygous mutation in the gene ZFHX1B (SIP1) [Cacheux et al., 2001]. In other patients, the correlation with the facial gestalt has not yet been clearly established [Amiel et al., 2001; Wakamatsu et al., 2001; Yamada et al., 2001]. Wehave recently seen aboywithHSCRwith the same phenotype as the cases described by Mowat et al. and having a mutation in the gene ZFHX1B (SIP1). The boy is the first of two children of healthy, nonconsanguineous parents (34-year-old mother and 34-year-old father). The mother’s sister had Noonan syndrome. Family history was otherwise unremarkable, none of the familymembers showedHSCR.Prenatalultrasound at 15 weeks gestation showed bilateral mild dilatation of the renal pelvis. There were poor fetal movements. Hewasbornat 40weeksgestation. Spontaneousvaginal vertex delivery occurred with a birth weight of 3,750 g, length of 51 cm, and head circumference of 34 cm. APGAR scores were 9 and 10 at one and five minutes, respectively. He was transferred from the Neonatal Service to pediatric surgery at the age of two days and underwent surgery for HSCR at the age of 50 days. He had hypotonia and global developmental delay, with involuntary nodding of the head and exaggerated reaction to acoustic stimulus. At the age of 17 months, he developed recurrent seizures, which responded to anticonvulsants, and partial epilepsy complex was diagnosed. On examination at the age of 18 months, his head circumference was 46 cm (10th centile), height was 83.5 cm (50th–75th centile), and weight was 10.16 kg (10th centile) He had fine and blond hair, high forehead, frontal bossing, prominent supraorbital ridges, deep set eyes, small nose with a bulbous tip, mouth held open, pointed chin, posterior angulation of ears, uplifted ear lobes, fetal finger pads, deep palmar and plantar creases, long halluces, hypospadias, and undescended right testis (Figs. 1 and 2). Dermatoglyphics showed 8/10 ulnar loops. Investigations including lactate, ammonium, QFQ banded karyotype at 650 band resolution, molecular analyses for fragile X syndrome, and flourescence in situ hybridization (FISH) for chromosome 8 mosaicism were all normal. Skeletal survey showed Wormian bones; hypoplasia of the distal phalanges of the fingers and hypoplasia of the middle and distal phalanges of the 2nd, 3rd, 4th, and 5th toes; and long and hypertrophic first ray of the feet. The patellae were normal. Cranial computed tomography (CT) demonstrated greater representation of subarachnoid spaces in frontal side compared to the remaining sites of the head, and magnetic resonance imaging (MRI) of the brain showed mild symmetrical accentuation of the apex of temporal horns bilaterally.Abdominal ultrasonographic evaluation was normal. Direct sequencing of ZFHX1B revealed a de novo heterozygous frameshift mutation nt901delC in exon 7, resulting in a stop codon after 36 aa. The truncated protein is missing part of the N-terminal zinc finger domain, the Smad binding domain, the homeodomain, and the C-terminal zinc finger domain (Fig. 3). The analysis was performed as described by Zweier et al. [2002]. *Correspondence to: Dr. Livia Garavelli, Divisione di Pediatria, Ambulatorio di Genetica Clinica, Arcispedale S. Maria NuovaAziendaOspedaliera-Viale Risorgimento, 80 42100 Reggio Emilia, Italy. E-mail: garavelli.livia@asmn.re.it

Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth

Sotos syndrome is characterized by pre‐ and post‐natal overgrowth, typical craniofacial features, advanced bone age, and developmental delay. Some degree of phenotypic overlap exists with other overgrowth syndromes, in particular with Weaver syndrome. Sotos syndrome is caused by haploinsufficiency of the NSD1 (nuclear receptor SET domain containing gene 1) gene. Microdeletions involving the gene are the major cause of the syndrome in Japanese patients, whereas intragenic mutations are more frequent in nonJapanese patients. NSD1 aberrations have also been described in some patients diagnosed as Weaver syndrome. Some authors have suggested a certain degree of genotype–phenotype correlation, with a milder degree of overgrowth, a more severe mental retardation, and a higher frequency of congenital anomalies in microdeleted patients. Data on larger series are needed to confirm this suggestion. We report here on microdeletion and mutation analysis of NSD1 in 59 patients with congenital overgrowth. Fourteen novel mutations, two previously described and one microdeletion were identified. All patients with a NSD1 mutation had been clinically classified as “classical Sotos,” although their phenotype analysis demonstrated that some major criteria, such as overgrowth and macrocephaly, could be absent. All patients with confirmed mutations shared the typical Sotos facial gestalt. A high frequency of congenital heart defects was present in patients with intragenic mutations, supporting the relevance of the NSD1 gene in the pathogenesis of this particular defect.

Editorial comment: Significance of linkage disequilibrium between the fragile X locus and its flanking markers

The identification of several microsatellite markers flanking the FRAXA locus was instrumental in the positional cloning of the FMR1 gene. These markers can still be valuable in family studies, e.g., as additional evidence in prenatal diagnosis. Additionally, they were employed to verify the presence of any significant gametic disequilibrium between the fragile X mutation and some haplotypes, although the high mutation rate predicted from early segregation studies implied that new mutants would arise on almost every chromosomal background. Thus, the discovery of linkage disequilibrium encompassing the fragile X locus has been surprising. Here, we review the available evidence of such gametic association and underline its implications for the mutational mechanism. 50 refs., 1 fig., 2 tabs.

A new case of interstitial deletion of chromosome 3q, del(3q)(q13.12q21.3), with agenesis of the corpus callosum.

We describe a boy with an interstitial deletion of the proximal portion of chromosome 3q. Prominent physical characteristics were a dysmorphic face with apparent hypertelorism, signs of prenatal lymphedema, foot contractures and agenesis of the corpus callosum. The finding of corpus callosum agenesis in the previously reported patient with an overlapping deletion suggests an additional locus for this malformation.

Constitutional trisomy 8 and myelodysplasia: Report of a case and review of the literature

A diagnosis of myelodysplastic syndrome was made in an 18-year-old patient with Warkany syndrome due to constitutional trisomy 8 mosaicism. The possible causal role of this particular chromosome constitution with respect to myelodysplasia and embryonal childhood tumors is discussed.

Two brothers with 22q13 deletion syndrome and features suggestive of the Clark-Baraitser syndrome.

We report on two brothers with moderate-to-severe mental retardation, severe macrocephaly, obesity, characteristic face, big hands and feet, advanced bone age and brain abnormalities, including frontal cortical atrophy. These two boys resembled the two brothers described by Clark and Baraitser (1987), two maternal cousins subsequently reported by Baraitser et al. (1995) and a Brazilian boy described by de Pina-Neto and Andreotti-de Molfetta (1998). Upon further investigation, we detected a cryptic subtelomeric deletion of chromosome region 22q13, not present in either parent and probably due to a maternal germinal mosaicism. Thus, we describe the first familial case of 22q13 deletion and recommend that patients with a phenotype suggestive of the so-called Clark–Baraitser syndrome be tested for submicroscopic 22qter deletion.

Cytogenetic findings in 4952 prenatal diagnoses. An Italian collaborative study

SummaryThe development of prenatal diagnosis in Italy was made difficult by the restrictions of the old abortion law and only in recent years has a consistent number of cases been investigated. We report the experience on prenatal chromosome diagnosis of ten Italian centers participating in a collaborative study on 4952 diagnoses performed from 1972 to 1980. The main indication groups were: advanced maternal age (2882 cases), previous child with chromosome anomaly from parents with normal karyotype (847 cases), and chromosome anomaly in one parent (97 cases). The other indications for amniocentesis, including cases without a cytogenetic risk, have been assembled into a “miscellaneous” group (1126 cases). We found 125 abnormal fetal karyotypes (2.5%) of which 89 were unbalanced (1.8%). The frequencies and types of chromosome anomalies are reported in detail for each indication group and are compared with the corresponding ones from the European Munich Conference. The great majority of these Italian data were not included in the Munich report.

The Simpson-Golabi-Behmel syndrome: a clinical case and a detective story

The Simpson–Golabi–Behmel syndrome (SGBS) is an overgrowth condition comprising “coarseness” of facial traits, supernumerary nipples, congenital heart defects, polydactyly and fingernail hypoplasia, and an increased risk of neonatal death and later neoplasia. Psychomotor development is usually normal. The syndrome is caused by mutation/deletion of the X‐linked gene GPC3. We describe a new case of SGBS, that led to the discovery of an extended family segregating a GPC3 mutation and, ultimately, of an affected relative forgotten, but not lost, in an anatomical museum, where he was classified as a macrosomic newborn, who was born probably around 1940 and died neonatally of unknown cause. This baby boy becomes the oldest case of SGBS on record.

Absence of 12q21.2q22 deletions and subtelomeric rearrangements in cardiofaciocutaneous (CFC) syndrome patients.

Recent publications described two patients with a CFC‐like phenotype and the same deletion of chromosome region 12q21.2q22 [Rauen et al., 2000 , 2002 ]. The patients did not have the classical CFC phenotype and presented other signs not usually seen in CFC patients: the first patient had hydrocephalus, and the second, a history of olygohydramnios, normal stature, pyloric stenosis, cutaneous syndactyly of toes and bilateral transverse palmar creases. In order to verify if classic CFC patients with normal chromosomes in conventional preparations have microdeletions within the 12q21.2q22 chromosome region, we performed FISH analysis using 12 BAC probes to screen this area. The average interval between the probes was of approximately 1 Mb. No deletions were found in any of the 17 classical CFC patients we examined. We conclude that the region 12q21.2q22 is not a candidate region for CFC syndrome and that the patients described by Rauen et al. [ 2000 , 2002 ] probably have a different condition, i.e., an aneuploidy syndrome, with some phenotypic resemblance to the CFC syndrome. To further evaluate the possibility of other chromosome imbalances, we performed a subtelomeric analysis, by FISH technique, of all chromosomes, and did not find any subtelomeric rearrangements.