Identity-by-descent and association mapping of a recessive gene for Hirschsprung disease on human chromosome 13q22

Hirschsprung disease (HSCR) is a congenital disorder of unknownetiology characterized by the absence of enteric ganglia Inthe distal colon. We have ascertained a large, inbred, Mennonitekindred which demonstrates a high Incidence of Hirschsprungdisease (HSCR). Genealogical analysis of all kinship relationshipsidentified a single common ancestral couple for all parentsof affected offspring. Segregation analysis yielded a segregationratio of 10.67% for males and 5.45% for females. We searchedfor locations of the gene(s) responsible for HSCR in this pedigreeby genotyplng three small multlcase families and locating genomlcregions demonstrating identity-by-descent followed by linkagedisequilibrium analysis of 28 additional nuclear families. Basedon this novel strategy, we report the mapping of a new locusfor HSCR to chromosome 13q22. Nine microsatelllte markers spanning10 cM in this region were genotyped on thirty-one nuclear families.Significant nonrandom association was detected with allelesat markers D13S162, D13S160, D13S170, and AFM240zg9. In addition,our studies reveal preliminary evidence for a genetic modifierof HSCR in this kindred on chromosome 21q22.     

Discordant phenotype of two overlapping deletions involving the PAX3 gene in chromosome 2q35

Waardenburg syndrome (WS), the most common form of Inheritedcongenltal deafness, is a pleiotropic, autosomal dominant conditionwith variable penetrance and expresslvity. WS is clinicallyand genetically heterogeneous. The basis for the phenotypicvariability observed among and between WS families is unknown.However, mutations within the paired-box gene, PAX3, have beenassociated with a subset of WS patients. In this report we usecytogenetic and molecular genetic techniques to study a patientwith WS type 3, a form of WS consisting of typical WS type 1features plus mental retardation, microcephaly, and severe skeletalanomalies. Our results show that the WS3 patient has a de novopaternally derived deletion, del (2)(q35q36), that spans thegenetic loci PAX3 and COL4A3. A molecular analysis of a chromosome2 deletional mapping panel maps the PAX3 locus to 2q35 and suggeststhe locus order: centromere-(INHA, DES)-PAX3-COL4A3-(ALPI, CHRND)-telomere.Our analyses also show that a patient with a cleft palate andlip pits, but lacking diagnostic WS features, has a deletion,del (2)(q33q35), Involving the PAX3 locus. This result suggeststhat not all PAX3 mutations are associated with a WS phenotypeand that additional regional loci may modify or regulate thePAX3 locus and/or the development of a WS phenotype.     

A novel susceptibility locus for Hirschsprung's disease maps to 4q31.3-q32.3

We report on a multigenerational family with isolated Hirschsprung's disease (HSCR). Five patients were affected by either short segment or long segment HSCR. The family consists of two main branches: one with four patients (three siblings and one maternal uncle) and one with one patient. Analysis of the RET gene, the major gene involved in HSCR susceptibility, revealed neither linkage nor mutations. A genome wide linkage analysis was performed, revealing suggestive linkage to a region on 4q31-q32 with a maximum parametric multipoint LOD score of 2.7. Furthermore, non-parametric linkage (NPL) analysis of the genome wide scan data revealed a NPL score of 2.54 (p = 0.003) for the same region on chromosome 4q (D4S413-D4S3351). The minimum linkage interval spans a region of 11.7 cM (12.2 Mb). No genes within this chromosomal interval have previously been implicated in HSCR. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome. Our results suggest the existence of a new susceptibility locus for HSCR at 4q31.3-q32.3.     

Interstitial deletion of 11q13 sequences in HeLa cells

Previous cytogenetic and molecular genetic studies have shown that the HeLa (cervical carcinoma) cell line D98/AH-2 contains two apparently normal copies of chromosome 11 and additional 11q13-25 material translocated onto a chromosome 3 marker. To determine the 11q13 breakpoint, we performed fluorescence in situ hybridization (FISH) using 18 different 11q13 specific BAC (bacterial artificial chromosome) and cosmid probes spanning a 5.6 Mb interval. Markers localized to the multiple endocrine neoplasia type 1 (MEN1) gene (menin) were also included in the analysis. The FISH study identified an interstitial deletion between markers D11S449 and GSTP1, an interval of 2.3 Mb, in the marker chromosome. This deletion did not include the MEN1 gene. Because point mutations and methylations can inactivate the MEN1 gene, single stranded conformational polymorphism (SSCP) and Northern and Western blot analyses were performed with MEN1 specific probes and antibody. SSCP did not reveal mutations of the MEN1 gene in HeLa or in seven other cervical cancer cell lines. Northern and Western blot studies revealed normal levels of expression of this gene in the cervical cancer cell lines as well as in HeLa cell derived tumorigenic hybrids. Because deletions of tumor suppressor genes often occur in cancer progression, we hypothesize that the inactivation of a tumor suppressor gene other than MEN1, localized to the 2.3 Mb interval on 11q13, might play a role in the abnormal growth behavior of HeLa cells in vitro or in vivo.     

Long segment and short segment familial Hirschsprung''s disease: variable clinical expression at the RET locus.

Hirschsprung's disease (aganglionic megacolon, HSCR) is a frequent condition of unknown origin (1/5000 live births) resulting in intestinal obstruction in neonates and severe constipation in infants and adults. In the majority of cases (80%), the aganglionic tract involves the rectum and the sigmoid colon only (short segment HSCR), while in 20% of cases it extends toward the proximal end of the colon (long segment HSCR). In a previous study, we mapped a gene for long segment familial HSCR to the proximal long arm of chromosome 10 (10q11.2). Further linkage analyses in familial HSCR have suggested tight linkage of the disease gene to the RET protoncogene mapped to chromosome 10q11.2. Recently, nonsense and missense mutations of RET have been identified in HSCR patients. However, the question of whether mutations of the RET gene account for both long segment and short segment familial HSCR remained unanswered. We have performed genetic linkage analyses in 11 long segment HSCR families and eight short segment HSCR families using microsatellite DNA markers of chromosome 10q. In both anatomical forms, tight pairwise linkage with no recombinant events was observed between the RET proto-oncogene locus and the disease locus (Zmax = 2.16 and Zmax = 5.38 for short segment and long segment HSCR respectively at 0 = 0%) Multipoint linkage analyses performed in the two groups showed that the maximum likelihood estimate was at the RET locus. Moreover, we show that point mutations of the RET proto-oncogene occur either in long segment or in short segment HSCR families and we provide evidence for incomplete penetrance of the disease causing mutation. These data suggest that the two anatomical forms of familial HSCR, which have been separated on the basis of clinical and genetic criteria, may be regarded as the variable clinical expression of mutations at the RET locus.     

Alveolar rhabdomyosarcoma with del(13q14)

We report a case of disseminated alveolar rhabdomyosarcoma, where chromosome analysis showed a deletion of chromosome 13(q14). This breakpoint is involved in the t(2;13)(q37;q14) previously reported in cases of rhabdomyosarcoma, but this is the first reported case in whom this deletion occurs without involvement of chromosome 2. The possible oncogenic role of the retinoblastoma (RB1) gene located at the breakpoint is discussed.     

Linkage analysis of genetic loci for kyphoscoliosis on chromosomes 5p13, 13q13.3, and 13q32

Kyphoscoliosis, a three-dimensional deformity of spinal growth, is characterized by a curvature in the coronal plane (scoliosis) in conjunction with thoracic kyphosis in excess of the normal range in the sagittal plane. We identified kyphoscoliosis within members of seven families (53 individuals) originally ascertained as part of a large collaborative study of familial idiopathic scoliosis. Model-independent linkage analysis of a genome-wide microsatellite screen identified areas suggestive of linkage on chromosomes 2q22, 5p13, 13q, and 17q11. Single-point and multipoint analyses of an additional 25 flanking microsatellite markers corroborated linkage to these regions, with areas on chromosomes 5p13, 13q13, and 13q32 being the most significant (P < 0.005). Analyses of single nucleotide polymorphism (SNP) markers in the candidate region on chromosome 5 narrowed the region to approximately 3.5 Mb (P < 0.05), with the most significant P values (P < 0.01) occurring in approximately a 1.3-Mb region. Candidate loci in this region include IRX1, IRX2, and IRX4 of the Iroquois Homeobox protein family. On chromosome 13, single-point and multipoint analyses resulted in multiple SNPs having P values < 0.05 within five candidate genes: Osteoblast-specific factor 2 or periostin, forkhead box O1A, A-kinase anchor protein 11, TBC1 domain family member 4, and glypican 5, thus supporting the potential relevance of this region in the pathogenesis of kyphoscoliosis.     

Isolated autosomal dominant type E brachydactyly: exclusion of linkage to candidate regions 2q37 and 20q13.

Type E brachydactyly is a digital malformation which characteristically causes an asymmetrical shortening of one or more metacarpals or metatarsals or both. Although commonly seen as part of a syndrome, it can be inherited as an autosomal dominant characteristic, the gene acting with variable expressivity, but complete penetrance. As an Albright hereditary osteodystrophy (AHO)-like syndrome including brachydactyly type E and mental retardation may be caused by (micro) deletions at chromosome 2q37, this region together with the AHO locus at chromosome 20q13 were considered as candidate loci for brachydactyly type E. In this paper we described a family with isolated autosomal dominant type E brachydactyly in whom molecular analysis excludes linkage to these regions, providing support for further genetic heterogeneity of this trait.     

Trimethylaminuria in a girl with Prader-Willi syndrome and del(15)(q11q13)

We report on an individual with trimethyl-aminuria, Prader-Willi syndrome, and del(15) (q11q13). To our knowledge, such an association has never been reported. Skin sores secondary to choline-rich foods and amenable to dietary control have not been described in trimethylaminuria, although they are seen in some patients with Prader-Willi syndrome. Pathogenesis, clinical diagnosis, and management of reported cases with trimethylaminuria are reviewed. Serious social and behavioral problems may result from strong body odor. Amelioration of the “fish odor” by dietary choline restriction makes trimethylaminuria detection important. Association of trimethylaminuria with Prader-Willi syndrome and del(15) (q11q13) in this patient is of particular interest. It may represent a contiguous gene syndrome, or deletion of the normal allele leading to expression of a single recessive trimethylaminuria gene, or an unrelated association, such as in Noonan syndrome. However, recent development of mapping of flavin-containing monooxygenase 2 (FMO2), the likely enzyme that is defective in fish odor syndrome, to chromosome 1q probably excludes pathogenetic association of fish odor syndrome with the Prader-Willi syndrome. © 1993 Wiley-Liss, Inc.     

Allelic loss on chromosome bands 13q11-q13 in esophageal squamous cell carcinoma.

Allelic loss on chromosome 13 occurs frequently in esophageal squamous cell carcinoma. However, studies of the two known tumor suppressor genes located on 13q, RB1 and BRCA2, have shown few mutations, suggesting that other genes are likely to be involved in the development of this tumor type. To identify a minimal deletion interval, we first analyzed 42 microsatellite markers spanning chromosome bands 13q11-q13 in 56 esophageal squamous cell carcinoma patients, including 34 with a family history of upper gastrointestinal cancer and 22 without a family history of cancer. Lifestyle risk factors and clinical/pathologic characteristics were also collected. Two commonly deleted regions were identified: one was located on band 13q12.11, between markers D13S787 and D13S221; the other was located on bands 13q12.3-q13.1 from markers D13S267 to D13S219. We observed higher allelic loss frequencies for eight of the microsatellite markers in those patients with a family history of upper gastrointestinal cancer compared to patients without such a history. This study suggests that one or more unidentified tumor suppressor genes are located on chromosome arm 13q that play a role in the development of esophageal squamous cell carcinoma.     

Identification of 13 novel mutations including a retrotransposal insertion in SLC25A13 gene and frequency of 30 mutations found in patients with citrin deficiency

Deficiency of citrin, liver-type mitochondrial aspartate-glutamate carrier, is an autosomal recessive disorder caused by mutations of the SLC25A13 gene on chromosome 7q21.3 and has two phenotypes: neonatal intrahepatic cholestatic hepatitis (NICCD) and adult-onset type II citrullinemia (CTLN2). So far, we have described 19 SLC25A13 mutations. Here, we report 13 novel SLC25A13 mutations (one insertion, two deletion, three splice site, two nonsense, and five missense) in patients with citrin deficiency from Japan, Israel, UK, and Czech Republic. Only R360X was detected in both Japanese and Caucasian. IVS16ins3kb identified in a Japanese CTLN2 family seems to be a retrotransposal insertion, as the inserted sequence (2,667-nt) showed an antisense strand of processed complementary DNA (cDNA) from a gene on chromosome 6 (C6orf68), and the repetitive sequence (17-nt) derived from SLC25A13 was found at both ends of the insert. All together, 30 different mutations found in 334 Japanese, 47 Chinese, 11 Korean, four Vietnamese and seven non-East Asian families have been summarized. In Japan, IVS16ins3kb was relatively frequent in 22 families, in addition to known mutations IVS11 + 1G > A, 851del4, IVS13 + 1G > A, and S225X in 189, 173, 48 and 30 families, respectively; 851del4 and IVS16ins3kb were found in all East Asian patients tested, suggesting that these mutations may have occurred very early in some area of East Asia.     

Association between high serum total IgE levels and D11S97 on chromosome 11q13 in Japanese subjects.

The genetic linkage of atopy to chromosome 11q13 through maternally derived alleles has been previously reported. Linkage analysis in Japanese families did not confirm the existence of a major gene for atopy at this locus under the model of autosomal dominant inheritance. However, we observed a significant association between serum total IgE levels and genetic markers at this locus both in 14 Japanese atopic families and in 120 unrelated Japanese subjects. We detected eight alleles at the D11S97 locus and eight alleles in the CA/GT repeat region in the fifth intron of the Fc epsilon RI beta gene. A significantly increased frequency of the D11S97/PstI 0.96 kb allele was observed in the chromosomes of the subjects with high serum total IgE levels both in the family study (p < 0.001) and in the population study (p < 0.05). However, multipoint linkage analysis again did not show any evidence for the existence of a major gene regulating atopy on chromosome 11q13 with location scores to -35 under the model of maternal inheritance. Evidence against linkage was confirmed by the non-parametric linkage analysis, using the affected pedigree member method. Also, there was no substitution of isoleucine for leucine in the fourth transmembrane domain of Fc epsilon RI beta (Leu181), which was reported to be responsible for a subset of atopy in the British population. Therefore, the association of serum total IgE levels with chromosome 11q13 indicates that a gene or genes at this locus may contribute to the expression of high IgE levels in the Japanese population as well as in the British population, but the heterogeneity of the genetic regulation of serum total IgE levels is evident between the two populations.     

Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22-q23.

We have identified six children with a distinctive facial phenotype in association with mental retardation (MR), microcephaly, and short stature, four of whom presented with Hirschsprung (HSCR) disease in the neonatal period. HSCR was diagnosed in a further child at the age of 3 years after investigation for severe chronic constipation and another child, identified as sharing the same facial phenotype, had chronic constipation, but did not have HSCR. One of our patients has an interstitial deletion of chromosome 2, del(2)(q21q23). These children strongly resemble the patient reported by Lurie et al with HSCR and dysmorphic features associated with del(2)(q22q23). All patients have been isolated cases, suggesting a contiguous gene syndrome or a dominant single gene disorder involving a locus for HSCR located at 2q22-q23. Review of published reports suggests that there is significant phenotypic and genetic heterogeneity within the group of patients with HSCR, MR, and microcephaly. In particular, our patients appear to have a separate disorder from Goldberg-Shprintzen syndrome, for which autosomal recessive inheritance has been proposed because of sib recurrence and consanguinity in some families.     

Endothelin-B receptor mutations in patients with isolated Hirschsprung disease from a non-inbred population

Hirschsprung disease (HSCR), or aganglionic megacolon, is the most common cause of congenital intestinal obstruction. Two different loci have been found to be tightly linked to HSCR on chromosomes 10 and 13, respectively. Recently, mutations in the RET protooncogene on chromosome 10q11.2 were identified in several HSCR patients. In addition, a missense mutation in the endothelin-B receptor (EDNRB) gene on chromosome 13q22 was found in an inbred Mennonite kindred affected by HSCR and associated abnormalities, demonstrating the involvement of EDNRB in HSCR pathogenesis. To test whether mutations in the EDNRB gene could account for Hirschsprung in patients from non-inbred populations, we analysed DNA samples from 17 probands of Italian origin with HSCR. We have identified two novel EDNRB mutations: a missense mutation in a sporadic case, S305N, which leads to a change of a serine to an asparagine, disrupting a putative phosphorylation site; and a single nucleotide deletion in a familial case, N378I, resulting in a truncated protein. Both mutations were found in one of the healthy parents, and neither of these mutations were found in any of the normal individuals tested. These data confirm the involvement of EDNRB in HSCR pathogenesis and demonstrate that EDNRB mutations could contribute to HSCR disease in non-inbred populations.      

Exclusion of RET and Pax 3 loci in Waardenburg-Hirschsprung disease.

The RET and the Pax 3 genes have recently been shown to account for autosomal dominant Hirschsprung's disease (HSCR) and Waardenburg syndrome type 1 (WS1) respectively, which led us to consider them as candidate genes in the WS/HSCR association. Linkage analyses performed in a consanguineous WS/HSCR family support the view that neither the RET locus nor the Pax 3 locus are involved in the disease phenotype. Hence, at least one further locus altering neural crest cell development is responsible for the pleiotropic features observed in the WS/HSCR association.     

Correlation between losses of IGH or its segments and deletions of 13q14 in t(11;14) (q13;q32) multiple myeloma

Multiple myeloma (MM) is a malignancy of the plasma cells (PCs) characterized by a wide variety of genetic and chromosomal abnormalities. In recent years, major attention was drawn to the significance of chromosomal aberrations involving chromosome arm 13q and the IGH region on chromosome band 14q32 as a prognostic indicator in MM. In this study we applied a combined cell morphology and FISH method for the analysis of coexistence of t(11;14)(q13;q32) with deletions of the long arm of chromosome 13 (Δ13) in PCs from 51 MM patients using several probes for the 13q14, 11q13, and IGH regions. We found 15 different variants of the t(11;14) that are the consequence of different 11q13 breakpoints and various deletions of Variable (del IGH Var) or Constant (del IGH Const) IGH segments and also duplications and losses of the IGH gene on the normal nontranslocated chromosome 14 as well as IGH/Cyclin D1 (CCND1) fusion on der(14) and CCND1/IGH fusions on der(11). A strong association between Δ13 and specific variants of t(11;14) was found: variants with deletion of the IGH gene or its segments were found only in MM cases with deleted chromosome 13, while the common translocation t(11;14) was found only in the MM cases with normal chromosome arm 13q. In contrast, we did not find any association between Δ13 and deletions of the IGH gene or its segments in the MM patients with t(4;14)(p16;q32). © 2009 Wiley‐Liss, Inc.     

Evidence for two independent associations with type 1 diabetes at the 12q13 locus

Genome-wide association studies have identified associations between type 1 diabetes and single-nucleotide polymorphisms (SNPs) at chromosome 12q13, surrounding the gene ERBB3. Our objective was to fine map this region to further localize causative variants. Re-sequencing identified more than 100 putative SNPs in an 80-kb region at 12q13. By genotyping 42 SNPs, spanning ～214?kb, in 382 affected sibling pair type 1 diabetes families, we were able to genotype or tag 67 common SNPs (MAF≥0.05) identified from HapMap CEU data and CEU data from the 1000 Genomes Project, plus additional rare coding variants identified from our re-sequencing efforts. In all, 15 SNPs provided nominal evidence for association (P≤0.05), with type 1 diabetes. The most significant associations were observed with rs2271189 (P=4.22 × 10(-5)), located in exon 27 of the ERBB3 gene, and an intergenic SNP rs11171747 (P=1.70 × 10(-4)). Follow-up genotyping of these SNPs in 2740 multiplex type 1 diabetes families validated these findings. After analyzing variants spanning more than 200?kb, we have replicated associations from previous GWAS and provide evidence for novel associations with type 1 diabetes. The associations across this region could be entirely accounted for by two common SNPs, rs2271189 and rs11171747.     

Mowat-Wilson syndrome

MWS is a multiple congenital anomaly syndrome, first clinically delineated by Mowat et al in 1998. Over 45 cases have now been reported. All patients have typical dysmorphic features in association with severe intellectual disability, and nearly all have microcephaly and seizures. Congenital anomalies, including Hirschsprung disease (HSCR), congenital heart disease, hypospadias, genitourinary anomalies, agenesis of the corpus callosum, and short stature are common. The syndrome is the result of heterozygous deletions or truncating mutations of the ZFHX1B (SIP1) gene on chromosome 2q22.     

Significant evidence for linkage of febrile seizures to chromosome 5q14-q15

Febrile seizures (FSs) represent the most common form of childhood seizure. In the Japanese population, the incidence rate is as high as 7%. It has been recognized that there is a significant genetic component for susceptibility to this type of seizure. Two putative FS loci, FEB1 (chromosome 8q13-q21) and FEB2 (chromosome 19p), have been mapped. Furthermore, a mutation in the voltage-gated sodium (Na(+))-channel beta1 subunit gene ( SCN1B ) at chromosome 19q13.1 was identified in a family with a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS(+)). These loci are linked to some large families. In this study, we conducted a genome-wide linkage search for FS in one large family with subsequent linkage confirmation in 39 nuclear families. Significant linkage was found at D5S644 by multipoint non-parametric analysis using GENEHUNTER ( P = 5.4 x 10(-6)). Estimated lambda(s)at D5S644 was 2.5 according to maximum likelihood analysis. Significant linkage disequilibria with FS were observed at the markers D5S644, D5S652 and D5S2079 in 47 families by transmission disequilibrium tests. These findings indicate that there is a gene on chromosome 5q14-q15 that confers susceptibility to FSs and we call this gene FEB4.